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PubMed2004

Site-specific localization of two distinct phosphatases along the osteoblast plasma membrane: tissue non-specific alkaline phosphatase and plasma membrane calcium ATPase.

In osteoblasts, alkaline phosphatase has been reported to be restricted to the basolateral domains. In recent studies, we have demonstrated phosphatase activities different from those of tissue non-specific alkaline phosphatase (TNSALP) along the osteoidal aspect of osteoblast membrane at alkaline and neutral pH on undecalcified freshly frozen sections of rat bones. In the present study, we sought to further characterize and define the nature of membrane-associated phosphatases along the osteoidal aspect of osteoblasts. Histochemical properties of the enzymes and their localization in vivo were examined in long bones of normal Wistar rats and TNSALP null mutant mice and their wild type littermates. Molecular profiles of the enzymes in the osteoblast extracts were also examined. The enzymatic activity of the phosphatase along the osteoidal surface of osteoblasts proved to be activated by both Mg2+ and Ca2+. Unlike TNSALP, the activity was inhibited by vanadate but resistant to levamisole, implicating a similarity between this enzyme and plasma membrane Ca2+ transport ATPase (PMCA). Immunohistochemistry showed that PMCA immunoreactions were restricted to the osteoidal domain of the plasma membrane. Native-PAGE analysis of osteoblast extracts suggested the presence of two phosphatases corresponding, respectively, to TNSALP and PMCA. Western blot analysis after SDS-PAGE of osteoblast extracts confirmed the existence of PMCA (140 kDa) and TNSALP (80 kDa). Gel-chemical analysis of the osteoblast extract from TNSALP null mutant mice depicted phosphatase activity, which was resistant to levamisole. These data suggest the presence of a phosphatase different from TNSALP, most plausibly PMCA, on the osteoidal surface of osteoblasts.

Alkaline Phosphatase↗
PubMed2003

Mechanism of the PII-activated phosphatase activity of Escherichia coli NRII (NtrB): how the different domains of NRII collaborate to act as a phosphatase.

The phosphatase activity of the homodimeric NRII protein of Escherichia coli is activated by the PII protein and requires all three domains of NRII. Mutations in the N-terminal domain (L16R), central domain (A129T), C-terminal domain PII-binding site (S227R), and C-terminal domain ATP-lid (Y302N) of NRII result in diminished phosphatase activity. Here, we used heterodimers formed in vitro from purified homodimeric proteins to study the phosphatase activity. A129T, S227R, and Y302N mutant subunits and A129T/S227R, A129T/Y302N, and S227R/Y302N double-mutant subunits formed stable heterodimers and were amenable to analysis; heterodimers containing these mutant subunits in various combinations were formed and their activities assessed. Complementation of the PII-activated phosphatase activity was observed in heterodimers containing S227R and Y302N subunits and in heterodimers containing A129T and Y302N subunits, but not in heterodimers containing A129T and S227R subunits. Complementation of the PII-activated phosphatase activity was also observed in heterodimers containing A129T/S227R and Y302N subunits, but not in heterodimers containing A129T/Y302N and S227R subunits. Finally, inclusion of an S227R/Y302N subunit in a heterodimer with a subunit having wild-type phosphatase activity resulted in a dramatic decrease in phosphatase activity, while inclusion of an A129T/S227R subunit did not. These results suggest that the phosphatase activity of NRII requires the collaboration of the PII-binding site from one subunit of the dimer, the central domain from the same subunit, and the ATP-lid from the opposing subunit, in addition to the undefined N-terminal domain requirement(s).

Binding Sites↗
PubMed2003

Myosin phosphatase-Rho interacting protein. A new member of the myosin phosphatase complex that directly binds RhoA.

Regulation of vascular smooth muscle cell contractile state is critical for the maintenance of blood vessel tone. Abnormal vascular smooth muscle cell contractility plays an important role in the pathogenesis of hypertension, blood vessel spasm, and atherosclerosis. Myosin phosphatase, the key enzyme controlling myosin light chain dephosphorylation, regulates smooth muscle cell contraction. Vasoconstrictor and vasodilator pathways inhibit and activate myosin phosphatase, respectively. G-protein-coupled receptor agonists can inhibit myosin phosphatase and cause smooth muscle cell contraction by activating RhoA/Rho kinase, whereas NO/cGMP can activate myosin phosphatase and cause smooth muscle cell relaxation by activation of cGMP-dependent protein kinase. We have used yeast two-hybrid screening to identify a 116-kDa human protein that interacts with both myosin phosphatase and RhoA. This myosin phosphatase-RhoA interacting protein, or M-RIP, is highly homologous to murine p116RIP3, is expressed in vascular smooth muscle, and is localized to actin myofilaments. M-RIP binds directly to the myosin binding subunit of myosin phosphatase in vivo in vascular smooth muscle cells by an interaction between coiled-coil and leucine zipper domains in the two proteins. An adjacent domain of M-RIP directly binds RhoA in a nucleotide-independent manner. M-RIP copurifies with RhoA and Rho kinase, colocalizes on actin stress fibers with RhoA and MBS, and is associated with Rho kinase activity in vascular smooth muscle cells. M-RIP can assemble a complex containing both RhoA and MBS, suggesting that M-RIP may play a role in myosin phosphatase regulation by RhoA.

Actin Cytoskeleton↗
PubMed2003

Relationship between phosphatase activity and cytotoxic effect of two protein phosphatase inhibitors, okadaic acid and pervanadate, on human myeloid leukemia cell line.

Protein phosphatases are signalling molecules that regulate a variety of fundamental cellular processes including cell growth, metabolism and apoptosis. The aim of this work was to correlate the cytotoxicity of pervanadate and okadaic acid on HL60 cells and their effect on the phosphatase obtained from these cells. The cytotoxicity of these protein phosphatase inhibitors was evaluated on HL60 cells using phosphatase activity, protein quantification and MTT reduction as indices. The major phosphatase presents in the cellular extract showed high activity (80%) and affinity (Km = 0.08 mM) to tyrosine phosphate in relation to p-nitrophenyl phosphate (pNPP)-(Km = 0.51 mM). Total phosphatase (pNPP) was inhibited in the presence of 10 mM vanadate (98%), 200 microM pervanadate (95%) and 100 microM p-chloromercuribenzoate (80%) but okadaic acid caused a slight increase in enzyme activity (25%). When the HL60 cells were treated with the phosphatase inhibitors (pervanadate and okadaic acid) for 24hours, only 20% residual activity was observed in presence of 200 microM pervanadate, whereas in the presence of okadaic acid this inhibitory effect was not observed. However, in respect to mitochondrial function, cell viability decreased about 80% in the presence of 100 nM okadaic acid. The total protein content was decreased 25% when the cells were treated with 100 nM okadaic acid in combination with 200 microM pervanadate. Our results suggest that both phosphatase inhibitors presented different mechanisms of action on HL60 cells. However, their effect on the cell redox status have to be considered.

Cell Survival↗
PubMed1985

Immunoalkaline phosphatase cytochemistry. Technical considerations of endogenous phosphatase activity.

The authors have developed an immunoalkaline phosphatase method and have applied it with success to the study of blood cells. They have now observed that macrophages in tissues and in serous effusions may be nonspecifically stained when immunoalkaline phosphatase methods are used. A systematic study of this endogenous macrophage phosphatase activity has shown it to have a pH optimum of 5.0-6.0 (acid phosphatase), but it remains weakly active in the mildly alkaline conditions used in the immunoalkaline phosphatase procedure. At its pH optimum, this macrophage phosphatase is mostly tartrate resistant, however, when 50 mM tartrate is added to a staining medium of pH 7.6-8.0, the residual endogenous phosphatase activity effectively is inhibited. When immunochemical studies are conducted by immunoalkaline phosphatase methods, the authors recommend addition of 50 mM tartrate to a buffer of pH 7.6-8.0. This modification does not significantly decrease the sensitivity of the specific staining of surface antigens.

Alkaline Phosphatase↗
PubMed1987

Serum osteocalcin, bone alkaline phosphatase isoenzyme and plasma tartrate resistant acid phosphatase in patients on chronic maintenance hemodialysis.

Biochemical indices of bone formation (serum osteocalcin and bone alkaline phosphatase isoenzyme) and osteoclastic function (plasma tartrate resistant acid phosphatase) were measured in 43 patients undergoing chronic hemodialysis and in 27 patients with primary hyperparathyroidism. The mean values for bone alkaline phosphatase isoenzyme and plasma tartrate resistant acid phosphatase but not for osteocalcin were significantly higher in primary hyperparathyroidism as compared with dialyzed patients. A significant positive correlation was found between the biochemical indices of osteoblasts and osteoclasts both in primary hyperparathyroidism and in dialyzed patients, indicating biological coupling between bone resorption and formation under these conditions. The regressions of osteocalcin vs bone alkaline phosphatase isoenzyme and/or plasma tartrate resistant acid phosphatase in dialyzed patients paralleled those in primary hyperparathyroidism but their distance differed significantly. It is concluded that in patients with renal failure, an increase in circulating osteocalcin by a relatively constant portion reflects decreased renal clearance. Any additional increase in osteocalcin serum level indicates an increased skeletal production of osteocalcin. The clinical value of bone alkaline phosphatase isoenzyme and plasma tartrate resistant acid phosphatase appears to be comparable with that of serum osteocalcin in primary hyperparathyroidism, and more exact than osteocalcin in renal failure.

Acid Phosphatase↗
PubMed1980

Cytochemistry and biochemistry of acid phosphatases. I. Cytochemistry and isoelectric focussing of acid phosphatases of the rat ventral prostate.

Acid phosphatases of the rat ventral prostate were studied cytochemically using different substrates. The results were compared to findings on isoelectric focussing gels stained for acid phosphatase activity. This is a highly specific and reproducible method which allows the distinction between secretory androgen-dependent and lysosomal acid phosphatases. Activity of lysosomal acid phosphatase was increased after castration, while the activity of the secretory enzyme gradually decreased after androgen deprivation. None of the substrates tested was selectively hydrolyzed by either secretory or lysosomal acid phosphatase. Phenylphosphate, creatine phosphate and choline phosphate were found to be inappropriate substrates for histochemical purposes, however, reproducible results were obtained with alpha-naphthylphosphate, beta-glycerophosphate and p-nitrophenylphosphate. The method of isoelectric focussing (pH range 4.0--8.0) of enzymes with subsequent histochemical staining demonstrated lysosomal enzymes at pH 7.9 and 8.2 respectively. Small amounts of identical enzymes were found in liver, kidney, blood or epididymis. Secretory acid phosphatases were focussed at pH 5.5, 5.6, 5.65 and 7.15. Similar enzymes have been identified in epididymis, kidney, liver and pancreas. These results indicate that 1) at present no "specific" substrate for prostatic secretory or lysosomal acid phosphatases is available and 2) that no prostate-specific "prostatic acid phosphatase (PAP)" exists in the rat ventral prostate.

Acid Phosphatase↗
PubMed1985

A major phosphotyrosyl-protein phosphatase from bovine heart is associated with a low-molecular-weight acid phosphatase.

The phosphotyrosyl [Tyr(P)]-immunoglobulin G (IgG) phosphatase activity in the extracts of bovine heart, bovine brain, human kidney, and rabbit liver can be separated by DEAE-cellulose at neutral pH into two fractions. The unbound fraction exhibits a higher activity at acidic than neutral pH while the reverse is true for the bound fraction. Of all tissues examined, the Tyr(P)-IgG phosphatase activity in the unbound fraction measured at pH 5.0 is higher than that in the bound fraction measured at pH 7.2. The acid Tyr(P)-IgG phosphatase activity has been extensively purified from bovine heart. It copurified with an acid phosphatase activity (p-nitrophenyl phosphate (PNPP) as a substrate) throughout the purification procedure. These two activities coelute from various ion-exchange and gel filtration chromatographies and comigrate on polyacrylamide gel electrophoresis, indicating that they reside on the same protein molecule. The phosphatase has a Mr = 15,000 by gel filtration and exhibits an optimum between pH 5.0 and 6.0 when either Tyr(P)-IgG-casein or PNPP is the substrate. It is highly specific for Tyr(P)-protein with little activities toward phosphoseryl [Ser(P)]- or phosphothreonyl [Thr(P)]-protein. The enzyme activities toward Tyr(P)-casein and PNPP are strongly inhibited by microM molybdate and vanadate but insensitive to inhibition by L(+)-tartrate, NaF, or Zn2+. The molecular and catalytic properties of the acid Tyr(P)-protein phosphatase purified from bovine heart are very similar to those of the low-molecular-weight acid phosphatases of Mr = 14,000 previously identified and purified from the cytosolic fraction of human liver, placenta, and other animal tissues.

Acid Phosphatase↗
PubMed1986

Protein phosphorylation and oocyte maturation. II. Inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases 1 and 2A and alkaline phosphatase.

Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. Microinjection of pure preparations of the catalytic subunits of protein phosphatases 1 and 2A inhibits 1-MeAde-induced maturation in a dose-dependent manner. Calmodulin-dependent protein phosphatase 2B is inefficient. Maturation induced by mimetics of 1-MeAde, such as dithiothreitol (DTT), methylglyoxal-bis(guanylhydrazone) (MGBG), 8-hydroxyeicosatetraenoic acid (8 HETE) and arachidonic acid (AA) is also inhibited by these protein phosphatases. In all cases inhibition can be reversed by increasing the concentration of 1-Me-Ade or of mimetic. Alkaline phosphatase also inhibits maturation in a dose-dependent way and in a reversible manner. Microinjection of protein phosphatase is still effective when preformed long after the end of the hormone-dependent period, and can even be effective a few minutes before the breakdown of the nuclear envelope. No detectable MPF activity is found in 1-MeAde-treated phosphatase-injected oocytes. However, microinjection of phosphatase 2A simultaneously with MPF (obtained from 1-MeAde-treated donors) does not result in inhibition. These results constitute direct evidence for the necessity of an elevated level of phosphorylated proteins for MPF activity and maturation. The mode of action of 1-MeAde in inducing starfish oocyte maturation is discussed in relation to protein phosphorylation.

Adenine↗
PubMed1991

The use of phosphopeptides to distinguish between protein phosphatase and acid/alkaline phosphatase activities: opposite specificity toward phosphoseryl/phosphothreonyl substrates.

The four main classes of protein phosphatases (PP-1, 2A, 2B and 2C), although differing in their ability to dephosphorylate phosphopeptide substrates, invariably display a marked preference toward phosphothreonyl peptides over their phosphoseryl counterparts. Conversely, all the acidic and alkaline phosphatases tested so far dephosphorylate phosphoseryl derivatives far more readily than phosphothreonyl ones. This opposite behaviour provides a criterion for discriminating between protein dephosphorylating activity due to authentic protein phosphatases as compared to nonspecific acid and/or alkaline phosphatases. In particular the phosphothreonyl peptides RRATPVA and RRREEETPEEEAA appear to be especially suited for detecting the activity of PP-2C and PP-2A, since they are hardly dephosphorylated by acid and alkaline phosphatases. Conversely, the phosphoseryl peptides SPEEEEE and RRASPVA can provide a sensitive evaluation of the majority of acid and alkaline phosphatases, while being refractory to protein phosphatases.

Acid Phosphatase↗
PubMed1997

Identification of a protein-tyrosine phosphatase (SHP1) different from that associated with acid phosphatase in rat prostate.

Using [32P]poly(Glu,Tyr) as substrate, we have identified, for the first time, in the rat prostatic gland a protein-tyrosine phosphatase activity different from that associated with prostatic acid phosphatase. Concanavalin A-Sepharose 4B was used to separate the two protein-tyrosyl phosphatases activities. The activity retained by the lectin had characteristics of the prostatic acid phosphatase. It was sensitive to inhibition by PNPP and the optimum pH shifted towards physiological values when [32P]poly(Glu,Tyr) was used as substrate. However, the major protein-tyrosine phosphatase activity was not retained by the lectin, and corresponded, at least in part, to SHP1 as probed by the presence of the protein, its mRNA and the loss of PTPase activity after immunodepletion of SHP1. This enzyme is localized within the epithelial cells. Thus, the coexistence of two protein-tyrosine phosphatase activities in rat prostate, one associated with the acid phosphatase and the other related to SHP1, makes it necessary to analyze the importance of both activities in vivo and their possible function regarding prostatic cell growth and its regulation.

Acid Phosphatase↗
PubMed1979

Purification and properties of a phosphorylase (phosphoprotein) phosphatase associated with an alkaline phosphatase of Mr 35000 from bovine adrenal cortex.

A metal-ion-independent, nonspecific phosphoprotein phosphatase (Mr = 35000) which represents the major phosphorylase phosphatase activity in bovine adrenal cortex has been purified to apparent homogeneity. An alkaline phosphatase activity (p-nitrophenyl phosphate as a substrate) of the same molecular weight, which requires both a metal ion (Mg2+ greater than Mn2+ greater than Co2+) and a sulfhydryl compound for activity, has been found to co-purify with the phosphoprotein phosphatase throughout the purification procedures. Characterization of the phosphoprotein and the alkaline phosphatase activities with respect to their catalytic properties, substrate and metal ion specificities, relationship with large molecular forms of the enzymes and responses to various effectors has been carried out. The results indicate that the phosphoprotein phosphatase can be converted by pyrophosphoryl compounds (e.g. PPi and ATP) to a metal-ion-dependent form which, subsequently, can be reactivated by Co2+ greater than Mn2+ but not by Mg2+ or Zn2+. The results also indicate that, although the phosphoprotein and the alkaline phosphatase activities are closely associated, they exhibit distinct physical and catalytic properties. Discussions concerning whether these two activities represent two different forms of the same protein or two different yet very similar polypeptide chains have been presented.

Adrenal Cortex↗
PubMed1999

Interactions of protein phosphatase type 1, with a focus on myosin phosphatase.

It has been established for many years that MLCK is regulated by the intracellular Ca2+ concentration via the formation of the Ca2+ -calmodulin-MLCK complex. A more recent discovery has been that the myosin phosphatase may also be regulated. This is manifest at suboptimal Ca2+ levels where under certain conditions (e.g. stimulation with several agonists) the MP is inhibited. The net result being that the extent of myosin phosphorylation for a fixed Ca2+ level is increased, i.e. an enhanced Ca2+ -sensitivity. Spurred by this intriguing discovery several laboratories began studies on MP with an emphasis to determine the regulatory, or inhibitory, mechanism. A similar preparation was obtained by 3 laboratories and consisted of a catalytic subunit, PP1delta, plus a large subunit (M130/133 for gizzard, M130 for bladder and M110 for rat aorta) and a smaller subunit of 20-21 kD. The isolated catalytic subunit has a much lower activity towards phosphorylated myosin than the holoenzyme, thus the non-catalytic subunits may serve as targeting proteins and in addition may play a regulatory role. Because of the difference in activities between the catalytic subunit and holoenzyme, one mechanism of regulation may involve dissociation of the trimeric complex, and such was proposed for the effect of arachidonic acid. Another suggested regulatory mechanism was that phosphorylation of the large subunit in its C-terminal half caused inhibition of phosphatase activity. The two mechanisms need not be mutually exclusive and in addition several kinases could influence the activity of the myosin phosphatase. In order to understand the molecular basis of phosphatase regulation it is necessary to determine the topography of the holoenzyme and identify sites of interaction between subunits and substrate. This work is in progress. Using various truncation mutants of M130/133 it has been determined that the binding sites for both PP1c and substrate are located within the N-terminal part of the molecule. The M20 subunit binds to the C-terminal end, although the functional significance of this is not established. Many questions remain to be answered concerning the biochemistry of the myosin phosphatase. An exciting and challenging focus will be to determine the mechanism(s) of regulation and to unravel the signaling cascade(s) that are initiated by agonist-receptor complex formation. In addition, the location of the MP is not known and it is important to establish which (if any) of the cytoskeletal elements are involved in binding to MP. Finally, it is assumed that the trimeric phosphatase, as discussed above, is specific for myosin dephosphorylation and does not act on other substrates. Because of the breadth of its distribution in different tissues and the wide range of proteins interacting with the ankyrin repeats it is possible that this phosphatase, or variants thereof, has roles in other cellular processes.

Animals↗
PubMed2000

Multiple forms of phosphatase from human brain: isolation and partial characterization of affi-gel blue binding phosphatases.

Implication of protein phosphatases in Alzheimer disease led us to a systemic investigation of the identification of these enzyme activities in human brain. Human brain phosphatases eluted from DEAE-Sephacel with 0.22 M NaCl were resolved into two main groups by affi-gel blue chromatography, namely affi-gel blue-binding phosphatases and affi-gel blue-nonbinding phosphatases. Affi-gel blue-binding phosphatases were further separated into four different phosphatases, designated P1, P2, P3, and P4 by calmodulin-Sepharose 4B and poly-(L-lysine)-agarose chromatographies. These four phosphatases exhibited activities towards nonprotein phosphoester and two of them, P1 and P4, could dephosphorylate phosphoproteins. The activities of the four phosphatases differed in pH optimum, divalent metal ion requirements, sensitivities to various inhibitors and substrate affinities. The apparent molecular masses as estimated by gel-filtration for P1, P2, P3, and P4 were 97, 45, 42, and 125 kDa, respectively. P1 is markedly similar to PP2B from bovine brain and rabbit skeletal muscle. P4 was labeled with anti-PP2A antibody and may represent a new subtype of PP2A. P1 and P4 were also effective in dephosphorylating Alzheimer disease abnormally hyperphosphorylated tau (AD P-tau). The resulting dephosphorylated AD P-tau had its activity restored in promoting assembly of microtubules in vitro. These results suggest that P1 and P4 might be involved in the regulation of phosphorylation of tau in human brain, especially in neurodegenerative conditions like Alzheimer's disease which are characterized by the abnormal hyperphosphorylation of this protein.

Alzheimer Disease↗
PubMed2000

Regulation of the DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol and growth phase. Inhibition of DGPP phosphatase activity by CDP-diacylglyceron and activation of phosphatidylserine synthase activity by DGPP.

The regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined. Addition of inositol to the growth medium resulted in a dose-dependent increase in the level of DGPP phosphatase activity in both exponential and stationary phase cells. Activity was greater in stationary phase cells when compared with exponential phase cells, and the inositol- and growth phase-dependent regulations of DGPP phosphatase were additive. Analyses of DGPP phosphatase mRNA and protein levels, and expression of beta-galactosidase activity driven by a P(DPP1)-lacZ reporter gene, indicated that a transcriptional mechanism was responsible for this regulation. Regulation of DGPP phosphatase by inositol and growth phase occurred in a manner that was opposite that of many phospholipid biosynthetic enzymes. Regulation of DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in opi1Delta, ino2Delta, and ino4Delta phospholipid synthesis regulatory mutants. CDP-diacylglycerol, a phospholipid pathway intermediate used for the synthesis of phosphatidylserine and phosphatidylinositol, inhibited DGPP phosphatase activity by a mixed mechanism that caused an increase in K(m) and a decrease in V(max). DGPP stimulated the activity of pure phosphatidylserine synthase by a mechanism that increased the affinity of the enzyme for its substrate CDP-diacylglycerol. Phospholipid composition analysis of a dpp1Delta mutant showed that DGPP phosphatase played a role in the regulation of phospholipid metabolism by inositol, as well as regulating the cellular levels of phosphatidylinositol.

Amino Acid Sequence↗
PubMed1988

DARPP-32 and phosphatase inhibitor-1, two structurally related inhibitors of protein phosphatase-1, are both present in striatonigral neurons.

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of Mr = 32,000) and phosphatase inhibitor-1, two previously characterized inhibitors of protein phosphatase-1, were identified in both the neostriatum and the substantia nigra. Phosphatase inhibitor-1 was partially purified from bovine caudate nucleus and found to be distinct from DARPP-32 in some of its biochemical properties. The neuronal localization of DARPP-32 and phosphatase inhibitor-1 within the rat neostriatum and substantia nigra was investigated by studying the effects of kainic acid. Injection into the neostriatum of kainic acid, which destroys striatonigral neurons and striatonigral fibers, decreased the amounts of DARPP-32 and phosphatase inhibitor-1 to the same extent, both in the lesioned neostriatum and in the ipsilateral substantia nigra. The specific activity of protein phosphatase-1 in the neostriatum was unaffected by kainic acid. The results indicate that, in rat brain, DARPP-32 and phosphatase inhibitor-1 are both present in striatal neurons and in striatonigral fibers, and that they probably coexist in at least a subpopulation of striatonigral neurons. In contrast, protein phosphatase-1 does not appear to be enriched in any specific neuronal subpopulation in the neostriatum.

Animals↗
PubMed1984

Brain dolichyl pyrophosphate phosphatase. Solubilization, characterization, and differentiation from dolichyl monophosphate phosphatase activity.

Dolichyl [beta-32P]pyrophosphate ([beta-32P]Dol-P-P) has been prepared chemically to study Dol-P-P phosphatase in calf brain. Calf brain microsomes catalyze the enzymatic release of 32Pi from exogenous [beta-32P]Dol-P-P by a bacitracin-sensitive reaction. [32P]Pyrophosphate was not detected with the water-soluble product even when 1 mM sodium pyrophosphate was added to impede pyrophosphatase activity. A substantial fraction of the Dol-P-P phosphatase activity can be solubilized by treating brain microsomes with 3% Triton X-100. The detergent extracts catalyze the enzymatic release of 32Pi from [beta-32P]Dol-P-P and the conversion of [14C]undecaprenyl pyrophosphate to [14C]undecaprenyl monophosphate. The solubilized Dol-P-P phosphatase activity: 1) is optimal at neutral pH; 2) is inhibited by Mn2+ and stimulated by EDTA; 3) exhibits an apparent Km = 20 microM for Dol-P-P; 4) is competitively inhibited by undecaprenyl pyrophosphate, and 5) is blocked by bacitracin. Solubilized Dol-P-P phosphatase activity differs from Dol-P phosphatase activity present in the same detergent extracts with respect to: 1) thermolability at 50 degrees C, 2) effect of 20 mM EDTA, and 3) sensitivity to phosphate and fluoride ions. These studies describe the chemical synthesis of [beta-32P]Dol-P-P for use in a convenient assay of Dol-P-P phosphatase activity. A procedure for the solubilization of Dol-P-P phosphatase activity from microsomes is presented, and an enzymological comparison indicates that Dol-P-P and Dol-P phosphatase are separate enzymes in calf brain.

Animals↗
PubMed1995

Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines.

BACKGROUND: Mitogen-activated protein (MAP) kinase is central to a signal transduction pathway that triggers cell proliferation or differentiation. Activation of the p42mapk isoform requires its phosphorylation at two residues, Thr 183 and Tyr 185, and this phosphorylation is catalysed by MAP kinase kinase (MAPKK). Relatively little is known, however, about the enzymes that dephosphorylate these residues, thereby inactivating the pathway. Recently, the CL100 phosphatase has been shown to inactivate p42mapk in vitro by dephosphorylating Thr 183 and Tyr 185 at similar rates. CL100, the product of an immediate early gene, is synthesized within one hour of stimulating cells with growth factors or exposure to oxidative stress or heat shock. Incubation of NIH 3T3 fibroblasts with cycloheximide prevents both synthesis of CL100 and inactivation of p42mapk after stimulation with serum. RESULTS: Depleting cells of CL100 and preventing its induction using cycloheximide stopped the inactivation of p42mapk in Swiss 3T3 fibroblasts following stimulation with epidermal growth factor (EGF), but had no effect on the rapid inactivation of p42mapk in response to EGF in adipose (3T3-L1) or chromaffin (PC12) cells or in response to platelet-derived growth factor (PDGF) in endothelial (PAE) cells. Moreover, maximal induction of CL100 mRNA and a CL100-like activity did not trigger inactivation of p42mapk, which was sustained at a high level after stimulation of PC12 cells with nerve growth factor, PAE cells with serum, or Swiss 3T3 cells with PDGF. Dephosphorylation of Tyr 185 but not Thr 183 of p42mapk was suppressed by vanadate in EGF-stimulated PC12 cells; dephosphorylation of Thr 183, by contrast, was elicited by a vanadate-insensitive activity. Protein phosphatase-2A was the only vanadate-insensitive phosphatase acting on Thr 183 of p42mapk or on MAPKK to be detected in PC12 cell extracts. Phosphorylation of Thr 183 also inhibited the dephosphorylation of Tyr 185 in vitro by the major vanadate-sensitive Tyr 185-specific phosphatase, explaining why dephosphorylation of Thr 183 is rate-limiting for p42mapk inactivation in PC12 cells after stimulation with EGF. CONCLUSIONS: The rapid inactivation of p42mapk initiated five minutes after stimulation of endothelial, adipose and chromaffin cells with growth factor is not catalysed by CL100, but rather by protein phosphatase 2A and by a protein tyrosine phosphatase distinct from CL100. Induction of CL100 is not accompanied by the inactivation of p42mapk in a number of situations.

3T3 Cells↗