Molecular cloning of the Escherichia coli gene for diadenosine 5'',5''''''-P1,P4-tetraphosphate pyrophosphohydrolase.

A clone overproducing diadenosine tetraphosphatase (diadenosine 5', 5'-P1, P4-tetraphosphate pyrophosphohydrolase) activity was isolated from an Escherichia coli cosmid library. Localization of the DNA region responsible for stimulation of this activity was achieved by deletion mapping and subcloning in various vectors. Maxicell experiments and immunological assays demonstrated that a 3.5-kilobase-pair DNA fragment carried the structural gene apaH encoding the E. coli diadenosine tetraphosphatase. The DNA coding strand was determined by cloning this fragment in both orientations in pUC plasmids. It was also shown that the overproduction of diadenosine tetraphosphatase decreased the dinucleoside tetraphosphate concentration in E. coli by a factor of 10.     

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

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 Asp608 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 Mg2+ or Mn2+, 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.     

Characterization of PrpC from Bacillus subtilis, a member of the PPM phosphatase family

We cloned the yloO gene and purified a His-tagged form of its product, the putative protein phosphatase YloO, which we now designate PrpC. This closely resembles the human protein phosphatase PP2C, a member of the PPM family, in sequence and predicted secondary structure. PrpC has phosphatase activity in vitro against a synthetic substrate, p-nitrophenol phosphate, and endogenous Bacillus subtilis proteins. The prkC and prpC genes are adjacent on the chromosome, and the phosphorylated form of PrkC is a substrate for PrpC. These findings suggest that PrkC and PrpC may function as a couple in vivo.     

Enzyme catalyzed hydrolysis of inorganic pyrophosphate. Current view of problems in characterization of PP1-phosphohydrolytic activity associated with alkaline phosphatases (EC 3.1.3.1).

A survey of the hydrolytic activity of alkaline phosphatase (EC 3.1.3.1) reveals that PP1, like phosphomonoesters, can serve as substrate in vitro. This pp1-phosphohydrolytic activity can be distinguished from PP1-phosphohydrolytic activities of inorganic pyrophosphatases (EC 3.6.1.1) and glucose-6-phosphatase (EC 3.1.3.9) by several criteria. Discrimination among these hydrolytic enzymes is possible by their dependence on variation of pH and of magnesium to PP1 ratios in the assay solutions. The true substrates and modifiers are not simply PP1 and magnesium, but the equilibrium species in mixtures of these two. The physiological significance of each of the three enzymes is not predictable from their differential efficiency as catalysts of PP1-hydrolysis in vitro.     

The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis

Reversible protein phosphorylation is critically important in the modulation of a wide variety of cellular functions. Several families of protein phosphatases remove phosphate groups placed on key cellular proteins by protein kinases. The complete genomic sequence of the model plant Arabidopsis permits a comprehensive survey of the phosphatases encoded by this organism. Several errors in the sequencing project gene models were found via analysis of predicted phosphatase coding sequences. Structural sequence probes from aligned and unaligned sequence models, and all-against-all BLAST searches, were used to identify 112 phosphatase catalytic subunit sequences, distributed among the serine (Ser)/threonine (Thr) phosphatases (STs) of the protein phosphatase P (PPP) family, STs of the protein phosphatase M (PPM) family (protein phosphatases 2C [PP2Cs] subfamily), protein tyrosine (Tyr) phosphatases (PTPs), low-M(r) protein Tyr phosphatases, and dual-specificity (Tyr and Ser/Thr) phosphatases (DSPs). The Arabidopsis genome contains an abundance of PP2Cs (69) and a dearth of PTPs (one). Eight sequences were identified as new protein phosphatase candidates: five dual-specificity phosphatases and three PP2Cs. We used phylogenetic analyses to infer clustering patterns reflecting sequence similarity and evolutionary ancestry. These clusters, particularly for the largely unexplored PP2C set, will be a rich source of material for plant biologists, allowing the systematic sampling of protein function by genetic and biochemical means.     

Arabidopsis PPP family of serine/threonine phosphatases

Serine/threonine-specific phosphoprotein phosphatases (PPPs) are ubiquitous enzymes in all eukaryotes, but their regulatory functions are largely unknown in higher plants. The Arabidopsis genome encodes 26 PPP catalytic subunits related to type 1, type 2A and so-called novel phosphatases, including four plant-specific enzymes carrying large N-terminal kelch-domains, but no apparent homologue of the PP2B family. The catalytic subunits of PPPs associate with regulatory protein partners that target them to well defined cellular locations and modulate their activity. Recent studies of phosphatase partners and their interactions have directed attention again to functional dissection of plant PPP families, and highlight their intriguing roles in the regulation of metabolism, cell cycle and development, as well as their roles in light, stress and hormonal signalling.     

The CAG repeat in SCA12 functions as a cis element to up-regulate PPP2R2B expression

PPP2R2B, a protein widely expressed in neurons, regulates the protein phosphatase 2A (PP2A) activity for dephosphorylation of tau and other substrates. CAG repeat expansion at the 5′-end of the PPP2R2B gene causes autosomal dominant spinocerebellar ataxia type 12. In the present study, we investigated the roles of CAG repeats and flanking cis elements and the associated proteins in controlling PPP2R2B expression. Deletion/site-directed mutagenesis, in silico searches and cDNA overexpression revealed that CREB1 and SP1 bind to the conserved sequence upstream the CAG repeats to up-regulate PPP2R2B expression, whereas TFAP4 binds to the conserved sequence downstream the CAG repeats to down-regulate PPP2R2B expression. The binding of CREB1, SP1, and TFAP4 to the PPP2R2B promoter was further confirmed by DNA pull-down and ChIP-PCR assays. CAG repeats itself also function as a cis element to up-regulate PPP2R2B expression as AT repeat length has no effect on PPP2R2B expression. Together, our data provide evidence that CREB1, SP1, and TFAP4 play roles in modulating PPP2R2B expression, thus offering a mechanism of regulating PP2A activity as the treatment of neurodegenerative diseases associated with abnormal PP2A activity.     

Insulin-stimulated phosphorylation of protein phosphatase 1 regulatory subunit 12B revealed by HPLC-ESI-MS/MS

ABSTRACT: BACKGROUND: Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. Protein phosphatase 1 regulatory subunit 12B (PPP1R12B), one of the regulatory subunits of PP1, can bind to PP1cdelta, one of the catalytic subunits of PP1, and modulate the specificity and activity of PP1cdelta against its substrates. Phosphorylation of PPP1R12B on threonine 646 by Rho kinase inhibits the activity of the PP1c-PPP1R12B complex. However, it is not currently known whether PPP1R12B phosphorylation at threonine 646 and other sites is regulated by insulin. We set out to identify phosphorylation sites in PPP1R12B and to quantify the effect of insulin on PPP1R12B phosphorylation by using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. RESULTS: 14 PPP1R12B phosphorylation sites were identified, 7 of which were previously unreported. Potential kinases were predicted for these sites. Furthermore, relative quantification of PPP1R12B phosphorylation sites for basal and insulin-treated samples was obtained by using peak area-based label-free mass spectrometry of fragment ions. The results indicate that insulin stimulates the phosphorylation of PPP1R12B significantly at serine 29 (3.02 +/- 0.94 fold), serine 504 (11.67 +/- 3.33 fold), and serine 645/threonine 646 (2.34 +/- 0.58 fold). CONCLUSION: PPP1R12B was identified as a phosphatase subunit that undergoes insulin-stimulated phosphorylation, suggesting that PPP1R12B might play a role in insulin signaling. This study also identified novel targets for future investigation of the regulation of PPP1R12B not only in insulin signaling in cell models, animal models, and in humans, but also in other signaling pathways.     

Phosphatase assay for multi-phosphorylated substrates using phosphatase specific-motif antibody

Protein phosphorylation plays central roles in a wide variety of signal transduction pathways and most phosphorylated proteins contain multi-phosphorylated sites. PPM1 type Ser/Thr protein phosphatase family is known to show rigid substrate specificity unlike other Ser/Thr phosphatase PPP family including PP1, PP2A and PP2B. PPM1 type phosphatases are reported to play important roles in growth regulation and in cellular stress signalling. In this study, we developed a phosphatase assay of PPM1D using phosphatase motif-specific antibody. PPM1D is a member of PPM1 type Ser/Thr phosphatase and known to dephosphorylate Ser(P)-Gln sequence. The gene amplification and overexpression of PPM1D were reported in many human cancers. We generated the monoclonal antibody specific for the Ser(P)-Gln sequence, named 3G9-H11. The specificity of this method using ELISA enables the convenient measurement of the dephosphorylation level of only PPM1D target residues of substrate peptides with multiple phosphorylated sites in the presence of multiple phosphatases. In addition, the antibody was applicable to immunoblotting assay for PPM1D function analysis. These results suggested that this method should be very useful for the PPM1D phosphatase assay, including high-throughput analysis and screening of specific inhibitors as anti-cancer drugs. The method using phosphatase motif-specific antibody can be applied to other PPM1 phosphatase family.     

Over expression of PPP2R2C inhibits human glioma cells growth through the suppression of mTOR pathway

PPP2R2C encodes a gamma isoform of the subunit B55 subfamily, which is a regulatory subunit of Protein phosphatase type 2A (PP2A). Our study shows that PPP2R2C is downregulated in glioma cells and human brain cancer patient samples. Overexpression of PPP2R2C inhibited cancer cell proliferation both in vitro and in vivo through the suppression of the activity of S6K in the mTOR pathway. Moreover, exogenous expression of PPP2R2C promoted the formation of a complex with the PP2A-C subunit to further enhance the binding of PP2A-C with S6K. Our results suggest that PPP2R2C is a potential tumor suppressor gene in human brain cancers. This study will provide novel insight into the development of therapeutic strategies in the treatment of human brain tumors.     

Disparate roles for the regulatory A subunit isoforms in Arabidopsis protein phosphatase 2A

The heterotrimeric protein phosphatase 2A (PP2A) complex comprises a catalytic subunit and regulatory A and B subunits that modulate enzyme activity and mediate interactions with other proteins. We report here the results of a systematic analysis of the Arabidopsis (Arabidopsis thaliana) regulatory A subunit gene family, which includes the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1), PP2AA2, and PP2AA3 genes. All three A subunit isoforms accumulate in the organs of seedlings and adult plants, suggesting extensive overlap in expression domains. We have isolated pp2aa2 and pp2aa3 mutants and found that their phenotypes are largely normal and do not resemble that of rcn1. Whereas rcn1 pp2aa2 and rcn1 pp2aa3 double mutants exhibit striking abnormalities in all stages of development, the pp2aa2 pp2aa3 double mutant shows only modest defects. Together, these data suggest that RCN1 performs a cardinal role in regulation of phosphatase activity and that PP2AA2 and PP2AA3 functions are unmasked only when RCN1 is absent.     

Mitochondrial location of rat liver dinucleoside triphosphatase

Rat liver dinucleoside triphosphatase (EC 3.6.1.29) is associated with sucrose-gradient purified mitochondria and can be extracted by freeze and thaw treatment. The proportion of mitochondrial dinucleoside triphosphatase approaches 50% of total liver enzyme. Evidence is also presented that 10% of total liver bis(5'-guanosyl)tetraphosphatase (EC 3.6.1.17) might be equally linked to mitochondria. Those data suggest that diadenosine 5',5'-P1,P3-triphosphate, diadenosine 5',5'-P1,P4-tetraphosphate, or other substrates of those enzymes, might be somehow related to mitochondria or mitochondrial function(s), although the occurrence of dinucleoside polyphosphates has not been reported in that organelle.     

Small molecule activators of pre-mRNA 3′ cleavage

3′ Cleavage and polyadenylation are obligatory steps in the biogenesis of most mammalian pre-mRNAs. In vitro reconstitution of the 3′ cleavage reaction from human cleavage factors requires high concentrations of creatine phosphate (CP), though how CP activates cleavage is not known. Previously, we proposed that CP might work by competitively inhibiting a cleavage-suppressing serine/threonine (S/T) phosphatase. Here we show that fluoride/EDTA, a general S/T phosphatase inhibitor, activates in vitro cleavage in place of CP. Subsequent testing of inhibitors specific for different S/T phosphatases showed that inhibitors of the PPM family of S/T phosphatases, which includes PP2C, but not the PPP family, which includes PP1, PP2A, and PP2B, activated 3′ cleavage in vitro. In particular, NCI 83633, an inhibitor of PP2C, activated extensive 3′ cleavage at a concentration 50-fold below that required by fluoride or CP. The testing of structural analogs led to the identification of a more potent compound that activated 3′ cleavage at 200 μM. While testing CP analogs to understand the origin of its cleavage activation effect, we found phosphocholine to be a more effective activator than CP. The minimal structural determinants of 3′ cleavage activation by phosphocholine were identified. Our results describe a much improved small molecule activator of in vitro pre-mRNA cleavage, identify the molecular determinants of cleavage activation by phosphoamines such as phosphocholine, and suggest that a PPM family phosphatase is involved in the negative regulation of mammalian pre-mRNA 3′ cleavage.     

PKR regulates B56(alpha)-mediated BCL2 phosphatase activity in acute lymphoblastic leukemia-derived REH cells

Protein phosphatase 2A (PP2A) is a heterotrimer comprising catalytic, scaffold, and regulatory (B) subunits. There are at least 21 B subunit family members. Thus PP2A is actually a family of enzymes defined by which B subunit is used. The B56 family member B56alpha is a phosphoprotein that regulates dephosphorylation of BCL2. The stress kinase PKR has been shown to phosphorylate B56alpha at serine 28 in vitro, but it has been unclear how PKR might regulate the BCL2 phosphatase. In the present study, PKR regulation of B56alpha in REH cells was examined, because these cells exhibit robust BCL2 phosphatase activity. PKR was found to be basally active in REH cells as would be predicted if the kinase supports B56alpha-mediated dephosphorylation of BCL2. Suppression of PKR promoted BCL2 phosphorylation with concomitant loss of B56alpha phosphorylation at serine 28 and inhibition of mitochondrial PP2A activity. PKR supports stress signaling in REH cells, as suppression of PKR promoted chemoresistance to etoposide. Suppression of PKR promoted B56alpha proteolysis, which could be blocked by a proteasome inhibitor. However, the mechanism by which PKR supports B56alpha protein does not involve PKR-mediated phosphorylation of the B subunit at serine 28 but may involve eIF2alpha activation of AKT. Phosphorylation of serine 28 by PKR promotes mitochondrial localization of B56alpha, because wild-type but not mutant S28A B56alpha promoted mitochondrial PP2A activity. Cells expressing wild-type B56alpha but not S28A B56alpha were sensitized to etoposide. These results suggest that PKR regulates B56alpha-mediated PP2A signaling in REH cells.     

Myosin phosphatase: Unexpected functions of a long-known enzyme

Myosin phosphatase (MP) holoenzyme is a Ser/Thr specific enzyme, which is the member of protein phosphatase type 1 (PP1) family and composed of a PP1 catalytic subunit (PP1c/PPP1CB) and a myosin phosphatase targeting subunit (MYPT1/PPP1R12A). PP1c is required for the catalytic activity of the holoenzyme, while MYPT1 regulates MP through targeting the holoenzyme to its substrates. Above the well-characterized function of MP, as the major regulator of smooth muscle contractility mediating the dephosphorylation of 20 kDa myosin light chain, accumulating data support its role in other, non-contractile functions. In this review, we summarize the scaffold function of MP holoenzyme and its roles in processes such as cell cycle, development, gene expression regulation and neurotransmitter release. In particular, we highlight novel interacting proteins of MYPT1 and pathophysiological functions of MP relevant to tumorigenesis, insulin resistance and neurodegenerative disorders.This article is part of a Special Issue entitled: Protein Phosphatases as Critical Regulators for Cellular Homeostasis edited by Prof. Peter Ruvolo and Dr. Veerle Janssens.