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PubMed1981

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

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

Alkaline Phosphatase↗
PubMed1981

Maximum activities and properties of glucose 6-phosphatase in muscles from vertebrates and invertebrates.

1. The maximum catalytic activities of glucose 6-phosphatase were measured in a large number of muscles from vertebrates and invertebrates. The activities range from less than 0.1 to 8.0 mumol/min per g fresh wt. at 30 degrees C: the highest activity, observed in the flight muscle of the wasp (Vespa vulgaris), is similar to that in rat liver. The hydrolytic activity was shown to be specific towards glucose 6-phosphate. 2. The pH optimum was 6.8 and the Km was approx. 0.6 mM (flight muscle of a moth). 3. Almost all of the glucose 6-phosphatase activity from extracts of the flight muscle of a moth and the pectoral muscle of a pigeon were recovered in the cytosolic fraction (i.e. 150,000 g supernatant). 4. During development of the locust (Schistocerca gregaria), the activity of the phosphatase in the flight muscle increased during the first 3 days after the final moult. 5. The activity of glucose 6-phosphatase from insect and avian muscle was separated from that of non-specific phosphatase on a Bio-Gel P-100 column. 6. For the activities from 63 muscles, there was a strong positive correlation between those of glucose 6-phosphatase and hexokinase, but no correlation between the activities of glucose 6-phosphatase and fructose bisphosphatase. It is suggested that the role of glucose 6-phosphate in muscle is either to produce glucose from glucose 6-phosphate derived from glycogen or to provide the enzymic basis for a substrate ("futile") cycle between glucose and glucose 6-phosphatase in muscle to improve the sensitivity of the mechanism that regulates the rate of glucose phosphorylation.

Animals↗
PubMed1983

Characterization of human foetal intestinal alkaline phosphatase. Comparison with the isoenzymes from the adult intestine and human tumour cell lines.

The molecular structure of human foetal intestinal alkaline phosphatase was defined by high-resolution two-dimensional polyacrylamide-gel electrophoresis and amino acid inhibition studies. Comparison was made with the adult form of intestinal alkaline phosphatase, as well as with alkaline phosphatases isolated from cultured foetal amnion cells (FL) and a human tumour cell line (KB). Two non-identical subunits were isolated from the foetal intestinal isoenzyme, one having same molecular weight and isoelectric point as placental alkaline phosphatase, and the other corresponding to a glycosylated subunit of the adult intestinal enzyme. The FL-cell and KB-cell alkaline phosphatases were also found to contain two subunits similar to those of the foetal intestinal isoenzyme. Characterization of neuraminidase digests of the non-placental subunit showed it to be indistinguishable from the subunits of the adult intestinal isoenzyme. This implies that no new phosphatase structural gene is involved in the transition from the expression of foetal to adult intestinal alkaline phosphatase, but that the molecular changes involve suppression of the placental subunit and loss of neuraminic acid from the non-placental subunit. Enzyme-inhibition studies demonstrated an intermediate response to the inhibitors tested for the foetal intestinal, FL-cell and KB-cell isoenzymes when compared with the placental, adult intestinal and liver forms. This result is consistent with the mixed-subunit structure observed for the former set of isoenzymes. In summary, this study has defined the molecular subunit structure of the foetal intestinal form of alkaline phosphatase and has demonstrated its expression in a human tumour cell line.

Alkaline Phosphatase↗
PubMed1985

Characterization of Ca2+-activated protein phosphatase activity in exocrine pancreas.

Ca2+-activated protein phosphatase activity was demonstrated in mouse pancreatic acinar cytosol with alpha-casein and skeletal-muscle phosphorylase kinase as substrates. This phosphatase activity preferentially dephosphorylated the alpha subunit of phosphorylase kinase. After DEAE-cellulose chromatography, the Ca2+-activated phosphatase activity became dependent on exogenous calmodulin for maximal activity. Half-maximal activation was achieved at 0.5 +/- 0.1 microM-Ca2+. Trifluoperazine completely inhibited Ca2+-activated phosphatase activity, with half-maximal inhibition occurring at 8.5 +/- 0.6 microM. Mn2+, but not Mg2+, at 1 mM concentration could substitute for Ca2+ in eliciting full enzyme activation. The apparent Mr of the phosphatase as determined by Sephadex G-150 chromatography was 93000 +/- 1000. Submitting active fractions obtained after Sephadex chromatography to calmodulin affinity chromatography resulted in the resolution of a major protein of Mr 55500 +/- 300. In conclusion, Ca2+-activated protein phosphatase activity has been identified in exocrine pancreas and has several features in common with Ca2+-activated calmodulin-dependent protein phosphatases previously isolated from brain and skeletal muscle. It is possible that this Ca2+-activated phosphatase may utilize as substrates certain acinar-cell phosphoproteins previously shown to undergo dephosphorylation in response to Ca2+-mediated secretagogues.

Animals↗
PubMed1986

Specificity of protein phosphatases in the dephosphorylation of protein kinase C.

Protein kinase C can autophosphorylate in vitro and has also been shown to be phosphorylated in vivo. In order to investigate the factors that may determine the phosphorylation state of protein kinase C in vivo, we determined the ability of the ATP + Mg2+-dependent phosphatase and the polycation-stimulated (PCS) phosphatases to dephosphorylate protein kinase C in vitro. These studies show that all the oligomeric forms of the PCS phosphatases (PCSH1, PCSH2, PCSM and PCSL phosphatases) are effective in the dephosphorylation of protein kinase C, showing 34-82% of the activity displayed with phosphorylase a as substrate. In contrast both the catalytic subunit of the PCS phosphatase and that of the ATP+Mg2+-dependent phosphatase showed only weak activity with protein kinase C as substrate. All these phosphatases, however, were activated by protamine (Ka 14-16 micrograms/ml) through what appears to be a substrate-directed effect. The relative role of these phosphatases in the control of protein kinase C is discussed.

Adenosine Triphosphate↗
PubMed1990

Polarized subcellular distribution of the 1-, 4- and 5-phosphatase activities that metabolize inositol 1,4,5-trisphosphate in intestinal epithelial cells.

In intestinal epithelial cells, Ins(1,4,5)P3 is metabolized both by an intracellular 5-phosphatase and by less specific extracellular phosphatases [Rubiera, Velasco, Michell, Lazo & Shears (1988) Biochem. J. 255, 131-137]. A total of 91% of intracellular Ins(1,4,5)P3 5-phosphatase was particulate, and was preferentially associated with plasma membranes rather than with other subcellular organelles. A soluble Ins(1,4,5)P3 3-kinase activity was also characterized, further supporting the idea that inositol phosphates are important in enterocyte function. We have studied the distribution of Ins(1,4,5)P3 phosphatase activities in basolateral and brush-border domains of the plasma membrane. Compared with homogenates, the extracellular phosphatases were 13-17-fold enriched in brush-border membranes, but only 2-fold enriched in basolateral membranes. The 1- and 4-phosphates of Ins(1,4,5)P3 were hydrolysed at equal rates by the extracellular phosphatases; these enzymes are proposed to have digestive functions. The intracellular particulate 5-phosphatase was 2-fold enriched in brush-border membranes and 13-fold enriched in basolateral membranes, at the same pole of the cell where Ins(1,4,5)P3 is believed to be generated. This is opposite to the polarized distribution of particulate 5-phosphatase in hepatocytes [Shears, Evans, Kirk & Michell (1988) Biochem. J. 256, 363-369]; these differences in subcellular distribution may be important in determining cell-specific metabolism of Ins(1,4,5)P3.

Animals↗
PubMed1993

Cloning and expression of the bovine intestinal alkaline phosphatase gene: biochemical characterization of the recombinant enzyme.

A complete genomic clone and a full-length cDNA coding for bovine intestinal alkaline phosphatase have been isolated and sequenced. The gene (5.4 kb) contains 11 exons separated by ten small introns at positions identical to those other members of the eukaryotic tissue-specific alkaline phosphatase family. In addition, 1.5 kb of upstream sequences contain putative regulatory elements showing sequence similarity to human and mouse intestinal alkaline phosphatase promoter sequences. To achieve recombinant bovine intestinal alkaline phosphatase expression, the coding region of the gene was subcloned into the pcDNA I eukaryotic expression vector and transfected into Chinese hamster ovary cells. Recombinant bovine intestinal alkaline phosphatase displays enzymatic properties comparable with those of purified native bovine intestinal alkaline phosphatase, a slightly increased thermal stability and, upon desialylation, it shows a homogeneous behaviour in agarose gel electrophoresis and isoelectric focusing. The availability of the recombinant bovine intestinal alkaline phosphatase and the elucidation of its primary sequence will help to accelerate our efforts to obtain the first crystallographic model of a eukaryotic alkaline phosphatase molecule.

Alkaline Phosphatase↗
PubMed2002

Can a determination of tartrate-resistant acid phosphatase predict postmenopausal loss of bone mass?

BACKGROUND: A study was carried out over a 24-month interval to determine if an initial measurement of serum tartrate-resistant acid phosphatase would be predictive of bone mass loss quantified by dual-energy X-ray absorptiometry, as total bone mineral content and total bone mineral content corrected for weight. DESIGN: Sixty-two women were studied (at onset: mean age 59.7 +/- 8.9 years, 10.8 +/- 8.8 years since menopause; at conclusion: mean age 61.9 +/- 8.8 and 13.0 +/- 8.7 since menopause). RESULTS: A paired Wilcoxon test showed a small, but significant, increase in weight (P < 0.05) and decrease in height (P < 0.05). Total bone mineral content and total bone mineral content corrected for weight decreased (P < 0.005 and 0.0001, respectively). Serum tartrate-resistant acid phosphatase increased (P < 0.005). Single-regression analysis showed that the per cent bone mass loss observed between the first and second body bone mineral content measurements correlated negatively with the first serum tartrate-resistant acid phosphatase determination (r = -0.62, P < 0.0001). Changes in tartrate-resistant acid phosphatase correlated negatively with changes in total bone mineral content (r = -0.79, P < 0.0001). In a multiple regression analysis of per cent change in bone mass against initially important variables such as age, years since menopause, weight, and tartrate-resistant acid phosphatase, only tartrate-resistant acid phosphatase was significant (P < 0.0001). The sensitivity and specifity of tartrate-resistant acid phosphatase for evaluating bone loss were 86% and 78%, respectively, and the area under the curve was of 0.83 (95% CI 0.71-0.95). CONCLUSION: These results show that a simple measurement of serum tartrate-resistant acid phosphatase can help to predict the potential rate of bone mass loss in women.

Absorptiometry, Photon↗
PubMed2006

Discovery of protein phosphatase inhibitor classes by biology-oriented synthesis.

Protein phosphatases have very recently emerged as important targets for chemical biology and medicinal chemistry research, and new phosphatase inhibitor classes are in high demand. The underlying frameworks of natural products represent the evolutionarily selected fractions of chemical space explored by nature so far and meet the criteria of relevance to nature and biological prevalidation most crucial to inhibitor development. We refer to synthesis efforts and compound collection development based on these criteria as biology-oriented synthesis. For the discovery of phosphatase inhibitor classes by means of this approach, four natural product-derived or -inspired medium-sized compound collections were synthesized and investigated for inhibition of the tyrosine phosphatases VE-PTP, Shp-2, PTP1B, MptpA, and MptpB and the dual-specificity phosphatases Cdc25A and VHR. The screen yielded four unprecedented and selective phosphatase inhibitor classes for four phosphatases with high hit rates. For VE-PTP and MptpB the first inhibitors were discovered. These results demonstrate that biology-oriented synthesis is an efficient approach to the discovery of new compound classes for medicinal chemistry and chemical biology research that opens up new opportunities for the study of phosphatases, which may lead to the development of new drug candidates.

Biological Products↗
PubMed1975

Inactivation of rabbit muscle phosphorylase phosphatase by cyclic AMP-dependent kinas.

Partially purified rabbit skeletal muscle phosphorylase phosphatase (EC 3.1.3.17; phosphoprotein phosphohydrolase) was inactivated when it was incubated with exogenous cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase), cyclic AMP, and ATP-Mg. Subsequent separation of the phosphatase by acrylamide gel electrophoresis or sucrose density centrifugation resulted in reactivation of the enzyme. The phosphatase decreased in molecular weight from approximately 70,000 to 52,000, and a phosphorylated inhibitor with molecular weight of 26,000 was found. Reactivation of phosphatase also occurred when it was incubated with MnCl2 or trypsin. The inhibitor was effective at less than 10(-8) M and was relatively heat stable. Its activity was destroyed by tryptic digestion and by dephosphorylation by a Mn-stimulated phosphatase. These observations support the possibility that phosphorylase phosphatase activity is controlled by cyclic AMP-dependent protein kinase and a Mn-stimulated phosphatase by a reaction involving phosphorylation and dephosphorylation of a protein phosphatase inhibitor.

Animals↗
PubMed1980

Expression of alkaline phosphatase loci in mammalian tissues.

Alkaline phosphatases [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] have been examined in liver, bone, kidney, intestine, and placenta from nine mammalian species by quantitative inhibition and thermostability studies and compared with alkaline phosphatases in the corresponding human tissues. In humans, three kinds of alkaline phosphatase can be sharply differentiated by these methods, one occurring in liver, bone, and kidney, one in intestine, and one in placenta. They are evidently determined by separate gene loci. In the mammals only two sorts of alkaline phosphatase were found: one, which occurs in liver, bone, kidney, and also placenta, corresponds to the human liver/bone/kidney enzyme and the other corresponds to the human intestinal enzyme. The findings support our earlier proposal that the expression of a distinctive type of alkaline phosphatase in human placenta is the consequence of a late evolutionary event which occurred subsequent to the divergence of the evolutionary lineage leading to humans from the various lineages leading to other mammalian species. The concentrations of the inhibitors, phenylalanine, homoarginine, phenylalanylglycylglycine, and levamisole, required to give 50% inhibition, [I(50)], of the liver/bone/kidney/placental (nonhuman) alkaline phosphatases showed no significant variation among the species. However, the [I(50)] values for the intestinal enzyme varied among species to a much greater extent. This implies that in the liver/bone/kidney/placental (nonhuman) alkaline phosphatase the structures of the binding sites for these inhibitors have been highly conserved during mammalian evolution, but there has been much greater divergence of these structures in the evolution of intestinal alkaline phosphatases.

Alkaline Phosphatase↗
PubMed1996

Purification and characterization of an RNA polymerase II phosphatase from yeast.

RNA polymerase (RNAP) II is subject to extensive phosphorylation on the heptapeptide repeats of the C-terminal domain (CTD) of the largest subunit. An activity that is required for the dephosphorylation of yeast RNAP II in vitro has been purified from a yeast whole cell extract by >30,000-fold. The yeast CTD phosphatase activity copurified with two bands with apparent molecular masses of 100 and 103 kDa. The properties of the yeast CTD phosphatase are similar to those of a previously characterized CTD phosphatase from HeLa cells. These properties include stimulation by the general transcription factor IIF (TFIIF), competitive inhibition by RNAP II, magnesium dependence, and resistance to okadaic acid. Both the HeLa and yeast CTD phosphatases are highly specific for their cognate polymerases. Neither phosphatase functions upon the polymerase molecule from the other species, even though the heptapeptide repeats of the CTDs in yeast RNAP II and mammalian RNAP II are essentially identical. The activity of the highly purified CTD phosphatase is stimulated >300-fold by a partially purified fraction of TFIIF. Recombinant TFIIF did not substitute for the TFIIF fraction, indicating that an additional factor present in the TFIIF fraction is required for CTD phosphatase activity. These results show that yeast contains a CTD phosphatase activity similar to that of mammalian cells that is likely composed of at least two components, one of which is 100 and/or 103 kDa.

Chromatography, Ion Exchange↗
PubMed1997

Isoprenylated human brain type I inositol 1,4,5-trisphosphate 5-phosphatase controls Ca2+ oscillations induced by ATP in Chinese hamster ovary cells.

D-myo-Inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase and 3-kinase are thought to be critical regulatory enzymes in the control of InsP3 and Ca2+ signaling. In brain and many other cells, type I InsP3 5-phosphatase is the major phosphatase that dephosphorylates InsP3 and D-myo-inositol 1,3,4,5-tetrakisphosphate. The type I 5-phosphatase appears to be associated with the particulate fraction of cell homogenates. Molecular cloning of the human brain enzyme identifies a C-terminal farnesylation site CVVQ. Post-translational modification of this enzyme promotes membrane interactions and changes in specific activity. We have now compared the cytosolic Ca2+ ([Ca2+]i) responses induced by ATP, thapsigargin, and ionomycin in Chinese hamster ovary (CHO-K1) cells transfected with the intact InsP3 5-phosphatase and with a mutant in which the C-terminal cysteine cannot be farnesylated. [Ca2+]i was also measured in cells transfected with an InsP3 3-kinase construct encoding the A isoform. The Ca2+ oscillations detected in the presence of 1 microM ATP in control cells were totally lost in 87.5% of intact (farnesylated) InsP3 5-phosphatase-transfected cells, while such a loss occurred in only 1.1% of the mutant InsP3 5-phosphatase-transfected cells. All cells overexpressing the InsP3 3-kinase also responded with an oscillatory pattern. However, in contrast to control cells, the [Ca2+]i returned to base-line levels in between a couple of oscillations. The [Ca2+]i responses to thapsigargin and ionomycin were identical for all cells. The four cell clones compared in this study also behaved similarly with respect to capacitative Ca2+ entry. In permeabilized cells, no differences in extent of InsP3-induced Ca2+ release nor in the threshold for InsP3 action were observed among the four clones and no differences in the expression levels of the various InsP3 receptor isoforms could be shown between the clones. Our data support the contention that the ATP-induced increase in InsP3 concentration in transfected CHO-K1 cells is essentially restricted to the site of its production near the plasma membrane, where it can be metabolized by the type I InsP3 5-phosphatase. This enzyme directly controls the [Ca2+]i response and the Ca2+ oscillations in intact cells.

Adenosine Triphosphate↗
PubMed1997

Kinetic and spectroscopic analyses of mutants of a conserved histidine in the metallophosphatases calcineurin and lambda protein phosphatase.

Calcineurin belongs to a family of serine/threonine protein phosphatases that contain active site dinuclear metal cofactors. Bacteriophage lambda protein phosphatase is also considered to be a member of this family based on sequence comparisons (Lohse, D. L., Denu, J. M., and Dixon, J. E. (1995) Structure 3, 987-990). Using EPR spectroscopy, we demonstrate that lambda protein phosphatase accommodates a dinuclear metal center. Calcineurin and lambda protein phosphatase likewise contain a conserved histidine that is not a metal ligand but is within 5 A of either metal in calcineurin. In this study the conserved histidine in calcineurin was mutated to glutamine and the mutant protein analyzed by EPR spectroscopy and kinetic methods. Parallel studies with an analogous lambda protein phosphatase mutant were also carried out. Kinetic studies using paranitrophenyl phosphate as substrate showed a decrease in kcat of 460- and 590-fold for the calcineurin and lambda protein phosphatase mutants, respectively, compared with the wild type enzymes. With a phosphopeptide substrate, mutagenesis of the conserved histidine resulted in a decrease in kcat of 1,300-fold for calcineurin. With the analogous lambda protein phosphatase mutant, kcat decreased 530-fold compared with wild type lambda protein phosphatase using phenyl phosphate as a substrate. EPR studies of the iron-reconstituted enzymes indicated that although both mutant enzymes can accommodate a dinuclear metal center, spectroscopic differences compared with wild type proteins suggest a perturbation of the ligand environment, possibly by disruption of a hydrogen bond between the histidine and a metal-coordinated solvent molecule.

Animals↗
PubMed1998

Distinct membrane and cytosolic forms of inositol polyphosphate 5-phosphatase II. Efficient membrane localization requires two discrete domains.

The 75-kDa inositol polyphosphate 5-phosphatase (5-phosphatase II) hydrolyzes various signaling molecules including the following: inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidylinositol 3,4, 5-trisphosphate. Although studied extensively, a demonstrably full-length cDNA encoding 5-phosphatase II has yet to be isolated. In this study we used a human partial 2.3-kilobase pair (kb) cDNA to screen mouse brain and kidney cDNA libraries, resulting in the isolation of a 3.7-kb cDNA (M5), which by multiple criteria represents a full-length cDNA encoding a 115-kDa 5-phosphatase II. We also isolated a smaller cDNA (M22) with a unique N terminus that encodes a 104-kDa polypeptide. Analysis of these cDNAs suggests a further 87-kDa isoform may arise from differential splicing resulting in translation at methionine 234 in M5. RNA analysis of tissues demonstrates expression of two mRNA species of approximately 4.0 or 3.0 kb, respectively. Probes unique to the 5' end of M5 or M22 hybridized to the 4.0- or 3.0-kb transcripts, respectively. RNA analysis using probes derived from sequence 3' to the potential splice site in M5 and M22 hybridized to both transcripts. Expression of the recombinant 115-kDa protein, or a smaller recombinant protein lacking the N terminus transiently in COS-7 cells, showed localization of enzyme activity to the membrane. Removal of the C-terminal CAAX motif resulted in a significant translocation of the protein lacking the N terminus but not the 115-kDa 5-phosphatase to the cytosol. Western blot analysis of membrane and cytosolic fractions of multiple mouse tissues confirmed the 115-kDa 5-phosphatase II was located in the membrane, whereas the 104- and 87-kDa isoforms were prominent in the cytosol. Collectively these studies demonstrate the widespread expression of at least three isoforms of 5-phosphatase II derived from RNA splicing events. This allows differential distribution of the 5-phosphatase II activity between the membrane and cytosol of the cell and thereby may regulate enzyme access to phosphoinositide-derived signaling molecules.

Amino Acid Sequence↗
PubMed1998

Regulation of carboxyl-terminal domain phosphatase by HIV-1 tat protein.

The phosphorylation state of the carboxyl-terminal domain (CTD) of RNA polymerase (RNAP) II is directly linked to the phase of transcription being carried out by the polymerase. Enzymes that affect CTD phosphorylation can thus play a major role in the regulation of transcription. A previously characterized HeLa CTD phosphatase has been shown to processively dephosphorylate RNAP II and to be stimulated by the 74-kDa subunit of TFIIF. This phosphatase is shown to be comprised of a single 150-kDa subunit by the reconstitution of catalytic activity from a SDS-polyacrylamide gel electrophoresis purified protein. This subunit has been previously cloned and shown to interact with the HIV Tat protein. To determine whether this interaction has functional consequences, the effect of Tat on CTD phosphatase was investigated. Full-length Tat-1 protein (Tat 86R) strongly inhibits the activity of CTD phosphatase. Point mutations in the activation domain of Tat 86R, which reduce the ability of Tat to transactivate in vivo, diminish its ability to inhibit CTD phosphatase. Furthermore, a deletion mutant missing most of the activation domain is unable to inhibit CTD phosphatase activity. The ability of Tat to transactivate in vitro also correlates with the strength of inhibition of CTD phosphatase. These results are consistent with the hypothesis that Tat-dependent suppression of CTD phosphatase is part of the transactivation function of Tat.

Gene Products, tat↗
PubMed2000

The inositol polyphosphate 5-phosphatases and the apurinic/apyrimidinic base excision repair endonucleases share a common mechanism for catalysis.

Inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from the inositol ring of phosphatidylinositol-derived signaling molecules; however, the mechanism of catalysis is only partially characterized. These enzymes play critical roles in regulating cell growth, apoptosis, intracellular calcium oscillations, and post-synaptic vesicular trafficking. The UCLA fold recognition server (threader) predicted that the conserved 300-amino acid catalytic domain, common to all 5-phosphatases, adopts the fold of the apurinic/apyrimidinic (AP) base excision repair endonucleases. PSI-BLAST searches of GENPEPT, using the amino acid sequence of AP endonuclease exonuclease III, identified all members of the 5-phosphatase family with highly significant scores. A sequence alignment between exonuclease III and all known 5-phosphatases revealed six highly conserved motifs containing residues that corresponded to the catalytic residues in the AP endonucleases. Mutation of each of these residues to alanine in the mammalian 43-kDa, or yeast Inp52p 5-phosphatase, resulted in complete loss of enzyme activity. We predict the 5-phosphatase enzymes share a similar mechanism of catalysis to the AP endonucleases, consistent with other common functional similarities such as an absolute requirement for magnesium for activity. Based on this analysis, functional roles have been assigned to conserved residues in all 5-phosphatase enzymes.

Amino Acid Sequence↗
PubMed2001

G-protein-coupled receptor stimulation of the p42/p44 mitogen-activated protein kinase pathway is attenuated by lipid phosphate phosphatases 1, 1a, and 2 in human embryonic kidney 293 cells.

Sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid bind to G-protein-coupled receptors to stimulate intracellular signaling in mammalian cells. Lipid phosphate phosphatases (1, 1a, 2, and 3) are a group of enzymes that catalyze de-phosphorylation of these lipid agonists. It has been proposed that the lipid phosphate phosphatases exhibit ecto activity that may function to limit bioavailability of these lipid agonists at their receptors. In this study, we show that the stimulation of the p42/p44 mitogen-activated protein kinase pathway by sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid, all of which bind to G(i/o)-coupled receptors, is substantially reduced in human embyronic kidney 293 cells transfected with lipid phosphate phosphatases 1, 1a, and 2 but not 3. This was correlated with reduced basal intracellular phosphatidic acid and not ecto lipid phosphate phosphatase activity. These findings were supported by results showing that lipid phosphate phosphatases 1, 1a, and 2 also abrogate the stimulation of p42/p44 mitogen-activated protein kinase by thrombin, a peptide G(i/o)-coupled receptor agonist whose bioavailability at its receptor is not subject to regulation by the phosphatases. Furthermore, the lipid phosphate phosphatases have no effect on the stimulation of p42/p44 mitogen-activated protein kinase by other agents that do not use G-proteins to signal, such as serum factors and phorbol ester. Therefore, these findings show that the lipid phosphate phosphatases 1, 1a, and 2 may function to perturb G-protein-coupled receptor signaling per se rather than limiting bioavailability of lipid agonists at their respective receptors.

Cell Line↗