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Mathieu Bollen - One of the best experts on this subject based on the ideXlab platform.
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Biogenesis and activity regulation of Protein Phosphatase 1
Biochemical Society Transactions, 2017Co-Authors: Iris Verbinnen, Mónica Ferreira, Mathieu BollenAbstract:Protein Phosphatase 1 (PP1) is expressed in all eukaryotic cells and catalyzes a substantial fraction of phosphoserine/threonine dephosphorylation reactions. It forms stable complexes with PP1-interacting Proteins (PIPs) that guide the Phosphatase throughout its life cycle and control its fate and function. The diversity of PIPs is huge (≈200 in vertebrates), and most of them combine short linear motifs to form large and unique interaction interfaces with PP1. Many PIPs have separate domains for PP1 anchoring, PP1 regulation, substrate recruitment and subcellular targeting, which enable them to direct associated PP1 to a specific subset of substrates and mediate acute activity control. Hence, PP1 functions as the catalytic subunit of a large number of multimeric holoenzymes, each with its own subset of substrates and mechanism(s) of regulation.
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Metals in the active site of native Protein Phosphatase-1.
Journal of Inorganic Biochemistry, 2015Co-Authors: Ewald Heroes, Monique Beullens, Luc Van Meervelt, Stefan De Gendt, Mathieu BollenAbstract:Protein Phosphatase-1 (PP1) is a major Protein Ser/Thr Phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed Phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone.
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Interactor-guided dephosphorylation by Protein Phosphatase-1
Methods of Molecular Biology, 2013Co-Authors: Shannah Boens, Aleyde Van Eynde, Kathelijne Szekér, Mathieu BollenAbstract:Protein Phosphatase-1 (PP1) is an essential enzyme for every eukaryotic cell and catalyzes more than half of all Protein dephosphorylations at serine and threonine residues. The free catalytic subunit of PP1 shows little substrate selectivity but is tightly regulated in vivo by a large variety of structurally unrelated PP1-interacting Proteins (PIPs). PIPs form highly specific dimeric or trimeric PP1 holoenzymes by acting as substrates, inhibitors, and/or substrate-specifiers. The surface of PP1 contains many binding sites for short PP1-docking motifs that are combined by PIPs to create a PP1-binding code that is universal, specific, degenerate, nonexclusive, and dynamic. These properties of the PP1-binding code can be used for the rational design of small molecules that disrupt subsets of PP1 holoenzymes and have a therapeutic potential.
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PPP1R8 (Protein Phosphatase 1, regulatory (inhibitor) subunit 8)
Atlas of genetics and cytogenetics in oncology and haematology, 2012Co-Authors: Nikki Minnebo, Monique Beullens, Mathieu BollenAbstract:Review on PPP1R8 (Protein Phosphatase 1, regulatory (inhibitor) subunit 8), with data on DNA, on the Protein encoded, and where the gene is implicated.
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Development of a peptide that selectively activates Protein Phosphatase-1 in living cells
Angewandte Chemie - International Edition, 2012Co-Authors: Jayanta Chatterjee, Monique Beullens, Rasa Sukackaite, Junbin Qian, Bart Lesage, Mathieu Bollen, Darren J. Hart, Maja KöhnAbstract:The first cell-penetrating peptide that activates Protein Phosphatase-1 (PP1) by disrupting a subset of PP1 complexes in living cells has been developed. Activated PP1 rapidly dephosphorylates its substrates, counteracting kinase activity inside cells. Activation of PP1 can thus be a novel approach to study PP1 function and to counteract Ser/Thr kinase activity under pathologically increased kinase signaling.
Paul Greengard - One of the best experts on this subject based on the ideXlab platform.
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phactrs 1 4 a family of Protein Phosphatase 1 and actin regulatory Proteins
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Patrick Allen, Audrey T Greenfield, Per Svenningsson, Derek C Haspeslagh, Paul GreengardAbstract:Protein Phosphatase 1 (PP1) is a multifunctional enzyme with diverse roles in the nervous system, including regulation of synaptic activity and dendritic morphology. PP1 activity is controlled via association with a family of regulatory subunits that govern subcellular localization and substrate specificity. A previously undescribed class of PP1-binding Proteins was detected by interaction cloning. Family members were also found to bind to cytoplasmic actin via Arg, Pro, Glu, and Leu repeat-containing sequences. The prototypical member of this family, Phosphatase and actin regulator (phactr) 1 was a potent modulator of PP1 activity in vitro. Phactr-1 Protein is selectively expressed in brain, where high levels were found in cortex, hippocampus, and striatum, with enrichment of the Protein at synapses. Additional family members displayed highly distinct mRNA transcript expression patterns within rat brain. The current findings present a mechanism by which PP1 may be directed toward neuronal substrates associated with the actin cytoskeleton.
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Preparation and characterization of recombinant Protein Phosphatase 1.
Methods in Enzymology, 2003Co-Authors: Takuo Watanabe, Paul Greengard, Hsien-bin Huang, Edgar F. Da Cruz E Silva, Natalia Starkova, Young-guen Kwon, Atsuko Horiuchi, Angus C. NairnAbstract:Publisher Summary The chapter describes preparation and characterization of recombinant Protein Phosphatase 1 (PP1). Various methods for preparation of PP1C in bacteria, and methods for preparation of PP1C in Sf9 cells using the baculovirus expression system are presented. The chapter discusses the preparation of phosphorylated and thiophosphorylated (dopamine- and cAMP-regulated phosphoProtein,M r 32,000)DARPP-32. Native PP1C is purified from rabbit skeletal muscle, using sequential chromatography on DEAE-cellulose, heparin-Sepharose, phenyl-Sepharose, Sephacryl S-200 and Mono-Q resins in order to recombinant PP1C. DARPP-32 and inhibitor-2 are prepared from E. coli as described. GST-tagged spinophilin and GSTPNUTS are prepared from E. coli as described. The chapter discusses Dephosphorylation of p-nitrophenyl phosphate (pNPP) and tyrosine-phosphorylated myelin basic Protein. The chapter describes two different procedures that are used to prepare recombinant PP1, using the baculovirus method. Several different affinity chromatography procedures are used that utilized the His6 and GST tags attached to recombinant PP1. The chapter discusses immunoblotting of recombinant PP1C.
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Protein Phosphatase 1 modulation of neostriatal ampa channels regulation by darpp 32 and spinophilin
Nature Neuroscience, 1999Co-Authors: Linda C Hsiehwilson, Angus C. Nairn, Jian Feng, Kazuhito Tomizawa, Patrick B Allen, Allen A Fienberg, Paul GreengardAbstract:Modulation of AMPA−type glutamate channels is important for synaptic plasticity. Here we provide physiological evidence that the activity of AMPA channels is regulated by Protein Phosphatase 1 (PP−1) in neostriatal neurons and identify two distinct molecular mechanisms of this regulation. One mechanism involves control of PP−1 catalytic activity by DARPP−32, a dopamine− and cAMP−regulated phosphoProtein highly enriched in neostriatum. The other involves binding of PP−1 to spinophilin, a Protein that colocalizes PP−1 with AMPA receptors in postsynaptic densities. The results suggest that regulation of anchored PP−1 is important for AMPA−receptor−mediated synaptic transmission and plasticity.
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a dopamine d1 receptor Protein kinase a dopamine and camp regulated phosphoProtein m r 32 kda Protein Phosphatase 1 pathway regulates dephosphorylation of the nmda receptor
The Journal of Neuroscience, 1998Co-Authors: Gretchen L Snyder, Allen A Fienberg, Richard L Huganir, Paul GreengardAbstract:We have investigated the mechanism by which activation of dopamine (DA) receptors regulates the glutamate sensitivity of medium spiny neurons of the nucleus accumbens. Our results demonstrate that DA regulates the phosphorylation state of the NR1 subunit of NMDA-type glutamate receptors. The effect of DA was mimicked by SKF82526, a D1-type DA receptor agonist, and by forskolin, an activator of cAMP-dependent Protein kinase (PKA), and was blocked by H-89, a PKA inhibitor. These data indicate that DA increases NR1 phosphorylation through a PKA-dependent pathway. DA-induced phosphorylation of NR1 was blocked in mice bearing a targeted deletion of the gene for dopamine- and cAMP-regulated phosphoProtein of M r 32 kDa (DARPP-32), a phosphoProtein that is a potent and selective inhibitor of Protein Phosphatase-1, indicating that the effect of PKA is mediated, in part, by regulation of the DARPP-32/Protein Phosphatase-1 cascade. In support of this interpretation, NR1 phosphorylation was increased by calyculin A, a Protein Phosphatase-1/2A inhibitor. A model is proposed in which the ability of DA to regulate NMDA receptor sensitivity is attributable to a synergistic action involving increased phosphorylation and decreased dephosphorylation of the NR1 subunit of the NMDA receptor.
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spinophilin a novel Protein Phosphatase 1 binding Protein localized to dendritic spines
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Patrick B Allen, Charles C Ouimet, Paul GreengardAbstract:Dendritic spines receive the vast majority of excitatory synaptic contacts in the mammalian brain and are presumed to contain machinery for the integration of various signal transduction pathways. Protein Phosphatase 1 (PP1) is greatly enriched in dendritic spines and has been implicated in both the regulation of ionic conductances and long-term synaptic plasticity. The molecular mechanism whereby PP1 is localized to spines is unknown. We have now characterized a novel Protein that forms a complex with the catalytic subunit of PP1 and is a potent modulator of PP1 enzymatic activity in vitro . Within the brain this Protein displays a remarkably distinct localization to the heads of dendritic spines and has therefore been named spinophilin. Spinophilin has the properties expected of a scaffolding Protein localized to the cell membrane and contains a single consensus sequence in PSD95/DLG/zo-1, which implies cross-linking of PP1 to transmembrane Protein complexes. We propose that spinophilin represents a novel targeting subunit for PP1, which directs the enzyme to those substrates in the dendritic spine compartment, e.g., neurotransmitter receptors, which mediate the regulation of synaptic function by PP1.
Monique Beullens - One of the best experts on this subject based on the ideXlab platform.
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Metals in the active site of native Protein Phosphatase-1.
Journal of Inorganic Biochemistry, 2015Co-Authors: Ewald Heroes, Monique Beullens, Luc Van Meervelt, Stefan De Gendt, Mathieu BollenAbstract:Protein Phosphatase-1 (PP1) is a major Protein Ser/Thr Phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed Phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone.
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PPP1R8 (Protein Phosphatase 1, regulatory (inhibitor) subunit 8)
Atlas of genetics and cytogenetics in oncology and haematology, 2012Co-Authors: Nikki Minnebo, Monique Beullens, Mathieu BollenAbstract:Review on PPP1R8 (Protein Phosphatase 1, regulatory (inhibitor) subunit 8), with data on DNA, on the Protein encoded, and where the gene is implicated.
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Development of a peptide that selectively activates Protein Phosphatase-1 in living cells
Angewandte Chemie - International Edition, 2012Co-Authors: Jayanta Chatterjee, Monique Beullens, Rasa Sukackaite, Junbin Qian, Bart Lesage, Mathieu Bollen, Darren J. Hart, Maja KöhnAbstract:The first cell-penetrating peptide that activates Protein Phosphatase-1 (PP1) by disrupting a subset of PP1 complexes in living cells has been developed. Activated PP1 rapidly dephosphorylates its substrates, counteracting kinase activity inside cells. Activation of PP1 can thus be a novel approach to study PP1 function and to counteract Ser/Thr kinase activity under pathologically increased kinase signaling.
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Protein Phosphatase 1 activates cdk9 by dephosphorylating ser175
PLOS ONE, 2011Co-Authors: Tatiana Ammosova, Monique Beullens, Mathieu Bollen, Yuri Obukhov, Alexander Kotelkin, Denitra Breuer, Victor R Gordeuk, Sergei NekhaiAbstract:The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity is regulated by Protein Phosphatase-1 (PP1) which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by (32P) in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D – inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription.
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Protein Phosphatase 1 regulates the histone code for long term memory
The Journal of Neuroscience, 2009Co-Authors: Kyoko Koshibu, Monique Beullens, Mathieu Bollen, Johannes Graff, Fabrice Heitz, Dominik Berchtold, Holger Russig, Melissa Farinelli, Isabelle M MansuyAbstract:Chromatin remodeling through histone posttranslational modifications (PTMs) and DNA methylation has recently been implicated in cognitive functions, but the mechanisms involved in such epigenetic regulation remain poorly understood. Here, we show that Protein Phosphatase 1 (PP1) is a critical regulator of chromatin remodeling in the mammalian brain that controls histone PTMs and gene transcription associated with long-term memory. Our data show that PP1 is present at the chromatin in brain cells and interacts with enzymes of the epigenetic machinery including HDAC1 (histone deacetylase 1) and histone demethylase JMJD2A (jumonji domain-containing Protein 2A). The selective inhibition of the nuclear pool of PP1 in forebrain neurons in transgenic mice is shown to induce several histone PTMs that include not only phosphorylation but also acetylation and methylation. These PTMs are residue-specific and occur at the promoter of genes important for memory formation like CREB (cAMP response element-binding Protein) and NF-κB (nuclear factor-κB). These histone PTMs further co-occur with selective binding of RNA polymerase II and altered gene transcription, and are associated with improved long-term memory for objects and space. Together, these findings reveal a novel mechanism for the epigenetic control of gene transcription and long-term memory in the adult brain that depends on PP1.
Angus C. Nairn - One of the best experts on this subject based on the ideXlab platform.
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structural basis for Protein Phosphatase 1 regulation and specificity
FEBS Journal, 2013Co-Authors: Wolfgang Peti, Angus C. Nairn, Rebecca PageAbstract:The ubiquitous serine/threonine Protein Phosphatase 1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, Protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. However, the free catalytic subunit of PP1, while an effective enzyme, lacks substrate specificity. Instead, it depends on a diverse set of regulatory Proteins (≥ 200) to confer specificity towards distinct substrates. Here, we discuss recent advances in structural studies of PP1 holoenzyme complexes and summarize the new insights these studies have provided into the molecular basis of PP1 regulation and specificity.
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Preparation and characterization of recombinant Protein Phosphatase 1.
Methods in Enzymology, 2003Co-Authors: Takuo Watanabe, Paul Greengard, Hsien-bin Huang, Edgar F. Da Cruz E Silva, Natalia Starkova, Young-guen Kwon, Atsuko Horiuchi, Angus C. NairnAbstract:Publisher Summary The chapter describes preparation and characterization of recombinant Protein Phosphatase 1 (PP1). Various methods for preparation of PP1C in bacteria, and methods for preparation of PP1C in Sf9 cells using the baculovirus expression system are presented. The chapter discusses the preparation of phosphorylated and thiophosphorylated (dopamine- and cAMP-regulated phosphoProtein,M r 32,000)DARPP-32. Native PP1C is purified from rabbit skeletal muscle, using sequential chromatography on DEAE-cellulose, heparin-Sepharose, phenyl-Sepharose, Sephacryl S-200 and Mono-Q resins in order to recombinant PP1C. DARPP-32 and inhibitor-2 are prepared from E. coli as described. GST-tagged spinophilin and GSTPNUTS are prepared from E. coli as described. The chapter discusses Dephosphorylation of p-nitrophenyl phosphate (pNPP) and tyrosine-phosphorylated myelin basic Protein. The chapter describes two different procedures that are used to prepare recombinant PP1, using the baculovirus method. Several different affinity chromatography procedures are used that utilized the His6 and GST tags attached to recombinant PP1. The chapter discusses immunoblotting of recombinant PP1C.
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Regulation of Protein Phosphatase-1
Chemistry & Biology, 2000Co-Authors: James B. Aggen, Angus C. Nairn, Richard ChamberlinAbstract:Abstract Reversible Protein phosphorylation is a major regulatory mechanism of intracellular signal transduction. Protein Phosphatase 1 (PP1) is one of four major types of serine-threonine Phosphatases mediating signaling pathways, but the means by which its activity is modulated has only recently begun to come into focus.
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Protein Phosphatase 1 modulation of neostriatal ampa channels regulation by darpp 32 and spinophilin
Nature Neuroscience, 1999Co-Authors: Linda C Hsiehwilson, Angus C. Nairn, Jian Feng, Kazuhito Tomizawa, Patrick B Allen, Allen A Fienberg, Paul GreengardAbstract:Modulation of AMPA−type glutamate channels is important for synaptic plasticity. Here we provide physiological evidence that the activity of AMPA channels is regulated by Protein Phosphatase 1 (PP−1) in neostriatal neurons and identify two distinct molecular mechanisms of this regulation. One mechanism involves control of PP−1 catalytic activity by DARPP−32, a dopamine− and cAMP−regulated phosphoProtein highly enriched in neostriatum. The other involves binding of PP−1 to spinophilin, a Protein that colocalizes PP−1 with AMPA receptors in postsynaptic densities. The results suggest that regulation of anchored PP−1 is important for AMPA−receptor−mediated synaptic transmission and plasticity.
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Phosphorylation and inactivation of Protein Phosphatase 1 by cyclin-dependent kinases
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: M Dohadwala, E F Da Cruz E Silva, Frederick L. Hall, Roger Williams, D. A. Carbonaro-hall, Angus C. Nairn, Paul Greengard, Norbert BerndtAbstract:Abstract Protein Phosphatase 1 and Protein Phosphatase 2A contain potential phosphorylation sites for cyclin-dependent kinases. In the present study we found that rabbit skeletal muscle Protein Phosphatase 1, as well as recombinant Protein Phosphatase 1 alpha and Protein Phosphatase 1 gamma 1, but not Protein Phosphatase 2A, was phosphorylated and inhibited by cdc2/cyclin A and cdc2/cyclin B. Phosphopeptide mapping and phospho amino acid analysis suggested that the phosphorylation site was located at a C-terminal threonine. Neither cdc2/cyclin A nor cdc2/cyclin B phosphorylated an active form of Protein Phosphatase 1 alpha in which Thr-320 had been mutated to alanine, indicating that the phosphorylation occurred at this threonine residue. Furthermore, Protein Phosphatase 1, but not Protein Phosphatase 2A, activity was found to change during the cell cycle of human MG-63 osteosarcoma cells. The observed oscillations in Protein Phosphatase 1 activity during the cell cycle may be due, at least in part, to phosphorylation of Protein Phosphatase 1 by cyclin-dependent kinases. Together, the results suggest a mechanism for direct regulation of Protein Phosphatase 1 activity.
Willy Stalmans - One of the best experts on this subject based on the ideXlab platform.
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The C-terminus of NIPP1 (nuclear inhibitor of Protein Phosphatase-1) contains a novel binding site for Protein Phosphatase-1 that is controlled by tyrosine phosphorylation and RNA binding.
The Biochemical journal, 2000Co-Authors: Monique Beullens, Willy Stalmans, A Van Eynde, V Vulsteke, I Jagiello, Mathieu BollenAbstract:Nuclear inhibitor of Protein Phosphatase-1 (NIPP1; 351 residues) is a nuclear RNA-binding Protein that also contains in its central domain two contiguous sites of interaction with the catalytic subunit of Protein Phosphatase-1 (PP1(C)). We show here that mutation of these Phosphatase-interaction sites did not completely abolish the ability of NIPP1 to bind and inhibit PP1(C). This could be accounted for by an additional inhibitory Phosphatase-binding site in the C-terminal region (residues 311-351), with an inhibitory core corresponding to residues 331-337. Following mutation of all three PP1(C)-binding sites in the central and C-terminal domains, NIPP1 no longer interacted with PP1(C). Remarkably, while both NIPP1 domains inhibited the phosphorylase Phosphatase activity of PP1(C) independently, mutation of either domain completely abolished the ability of NIPP1 to inhibit the dephosphorylation of myelin basic Protein. The inhibitory potency of the C-terminal site of NIPP1 was decreased by phosphorylation of Tyr-335 and by the addition of RNA. Tyr-335 could be phosphorylated by tyrosine kinase Lyn, but only in the presence of RNA. In conclusion, NIPP1 contains two Phosphatase-binding domains that function co-operatively but which are controlled independently. Our data are in agreement with a shared-site model for the interaction of PP1(C) with its regulatory subunits.
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The C-terminus of NIPP1 (nuclear inhibitor of Protein Phosphatase-1) contains a novel binding site for Protein Phosphatase-1 that is controlled by tyrosine phosphorylation and RNA binding
Biochemical Journal, 2000Co-Authors: Monique Beullens, Willy Stalmans, Veerle Vulsteke, Izabela Jagiello, Aleyde Van Eynde, Mathieu BollenAbstract:Nuclear inhibitor of Protein Phosphatase-1 (NIPP1; 351 residues) is a nuclear RNA-binding Protein that also contains in its central domain two contiguous sites of interaction with the catalytic subunit of Protein Phosphatase-1 (PP1 C ). We show here that mutation of these Phosphatase-interaction sites did not completely abolish the ability of NIPP1 to bind and inhibit PP1 C . This could be accounted for by an additional inhibitory Phosphatase-binding site in the C-terminal region (residues 311‐351), with an inhibitory core corresponding to residues 331‐337. Following mutation of all three PP1 C -binding sites in the central and C-terminal domains, NIPP1 no longer interacted with PP1 C . Remarkably, while both NIPP1 domains inhibited the phosphorylase Phosphatase activity of PP1 C independently, mutation
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IDENTIFICATION OF MYPT1 AND NIPP1 AS SUBUNITS OF Protein Phosphatase 1 IN RAT LIVER CYTOSOL
FEBS Letters, 1999Co-Authors: An Boudrez, Willy Stalmans, Monique Beullens, Etienne Waelkens, Kristien Evens, Mathieu BollenAbstract:Various studies have provided evidence for the existence of spontaneously active cytosolic species of Protein Phosphatase 1, but these enzymes have never been purified and characterized. We have used chromatography on microcystin-Sepharose and Resource Q to purify cytosolic Protein Phosphatases from rat liver. Two of the isolated enzymes were identified by Western analysis and peptide sequencing as complexes of the catalytic subunit of Protein Phosphatase 1 and either the inhibitor NIPP1 or the myosin-binding subunit MYPT1, which reportedly is not present in chicken liver. In contrast, PCR cloning revealed the expression of two MYPT1 splice variants in rat liver.
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Structure and splice products of the human gene encoding sds22, a putative mitotic regulator of Protein Phosphatase-1
European Journal of Biochemistry, 1999Co-Authors: Hugo Ceulemans, Aleyde Van Eynde, Encarna P??rez-callej??n, Willy Stalmans, Monique Beullens, Mathieu BollenAbstract:sds22 is a regulatory subunit of Protein Phosphatase-1 that is required for the completion of mitosis in yeast. It consists largely of 11 tandem leucine-rich repeats of 22 residues that are expected to mediate interactions with other polypeptides, including Protein Phosphatase-1. In this paper, we report on the structure of the human gene encoding sds22, designated PPP1R7. This gene (33 kb) comprises 11 exons, but these do not coincide with the sequences encoding the leucine-rich repeats. Up to six splice variants can be generated by exon skipping and alternative polyadenylation, as revealed by expressed sequence tag database analysis, RT-PCR and Northern blot analysis. The sds22 transcripts are expected to encode four different polypeptides. sds22alpha1 corresponds to the variant cloned previously from human brain [Renouf et al. (1995) FEBS Lett. 375, 75-78]. Sds22beta1 is truncated within the ninth repeat and has a short and different C-terminus. Both variants also exist without the sequence corresponding to exon 2, and these are termed sds22alpha2 and sds22beta2. The 5'-flanking region of PPP1R7 contains two NF-Y-binding CCAAT boxes near the transcription start site and potential binding sites for the transcription factors c-Myb, Ik-2 and NF-1, which are conserved in the mouse gene.
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Properties and Phosphorylation Sites of Baculovirus-expressed Nuclear Inhibitor of Protein Phosphatase-1 (NIPP-1)
Journal of Biological Chemistry, 1997Co-Authors: Veerle Vulsteke, Willy Stalmans, Monique Beullens, Etienne Waelkens, Mathieu BollenAbstract:Abstract NIPP-1 is the RNA-binding subunit of a major species of Protein Phosphatase-1 in the nucleus. We have expressed nuclear inhibitor of Protein Phosphatase-1 (NIPP-1) in Sf9 cells, using the baculovirus-expression system. The purified recombinant Protein was a potent (K i = 9.9 ± 0.3 pm) and specific inhibitor of Protein Phosphatase-1 and was stoichiometrically phosphorylated by Protein kinases A and CK2. At physiological ionic strength, phosphorylation by these Protein kinases drastically decreased the inhibitory potency of free NIPP-1. Phosphorylation of NIPP-1 in a heterodimeric complex with the catalytic subunit of Protein Phosphatase-1 resulted in an activation of the holoenzyme without a release of NIPP-1. Sequencing and phosphoamino acid analysis of tryptic phosphopeptides enabled us to identify Ser178 and Ser199 as the phosphorylation sites of Protein kinase A, whereas Thr161 and Ser204were phosphorylated by Protein kinase CK2. These residues all conform to consensus recognition sites for phosphorylation by Protein kinases A or CK2 and are clustered near a RVXF sequence that has been identified as a motif that interacts with the catalytic subunit of Protein Phosphatase-1.
