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PubMed1997

Role of oxidative stress in the action of vanadium phosphotyrosine phosphatase inhibitors. Redox independent activation of NF-kappaB.

The role of intracellular oxidative stress in the mechanism of action of phosphotyrosine phosphatase (PTP) inhibitors was studied using three vanadium-based compounds. Sodium orthovanadate (Na3VO4), sodium oxodiperoxo(1,10-phenanthroline)vanadate(V) (pV(phen), and bis(maltolato)-oxovanadium(IV) (BMOV) differentially induced oxidative stress in lymphocytes. Treatment with pV(phen), which caused intracellular oxidation, induced strong protein tyrosine phosphorylation compared with Na3VO4 and BMOV. Syk family kinases and the mitogen-activated protein kinase erk2 were rapidly activated by pV(phen) but not by BMOV or Na3VO4. In contrast, both BMOV and pV(phen) strongly activated NF-kappaB. The antioxidant pyrrolidine dithiocarbamate (PDTC) greatly diminished the intracellular oxidation and protein phosphotyrosine accumulation induced by pV(phen). Pretreatment of cells with PDTC reduced and delayed the activation of Syk kinases and erk2. However, NF-kappaB activation by pV(phen) was markedly enhanced in lymphocytes pretreated with PDTC, and another antioxidant, N-acetylcysteine, did not prevent the activation of NF-kappaB by BMOV. These results indicate a role for oxidative stress in the biological effects of some PTP inhibitors, whereas NF-kappaB activation by PTP inhibitors is mediated by mechanisms independent of intracellular redox status.

Antioxidants↗
PubMed1997

Antisense inhibition of group VI Ca2+-independent phospholipase A2 blocks phospholipid fatty acid remodeling in murine P388D1 macrophages.

A major issue in lipid signaling relates to the role of particular phospholipase A2 isoforms in mediating receptor-triggered responses. This has been difficult to study because of the lack of isoform-specific inhibitors. Based on the use of the Group VI Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL), we previously suggested a role for the iPLA2 in mediating phospholipid fatty acid turnover (Balsinde, J., Bianco, I. D., Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92: 8527-8531). We have now further evaluated the role of the iPLA2 in phospholipid remodeling by using antisense RNA technology. We show herein that inhibition of iPLA2 expression by a specific antisense oligonucleotide decreases both the steady-state levels of lysophosphatidylcholine and the capacity of the cell to incorporate arachidonic acid into membrane phospholipids. These effects correlate with a decrease in both iPLA2 activity and protein in the antisense-treated cells. Collectively these data provide further evidence that the iPLA2 plays a major role in regulating phospholipid fatty acyl turnover in P388D1 macrophages. In stark contrast, experiments with activated cells confirmed that the iPLA2 does not play a significant role in receptor-coupled arachidonate mobilization in these cells, as manifested by the lack of an effect of the iPLA2 antisense oligonucleotide on PAF-stimulated arachidonate release.

Animals↗
PubMed1998

UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway that is associated with inactivation of the Wee1Hu kinase and activation of the Cdc25C phosphatase.

We have previously demonstrated that UCN-01, a potent protein kinase inhibitor currently in phase I clinical trials for cancer treatment, abrogates G2 arrest following DNA damage. Here we used murine FT210 cells, which contain temperature-sensitive Cdc2 mutations, to determine if UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway. We report that UCN-01 cannot induce mitosis in DNA-damaged FT210 cells at the non-permissive temperature for Cdc2 function. Failure to abrogate G2 arrest was not due to UCN-01-inactivation at the elevated temperature because parental FM3A cells, which have wild-type Cdc2, were sensitive to UCN-01-induced G2 checkpoint abrogation. Having established that UCN-01 acted through Cdc2, we next assessed UCN-01's effect on the Cdc2-inhibitory kinase, Wee1Hu, and the Cdc2-activating phosphatase, Cdc25C. We found that Wee1Hu was indeed inactivated in UCN-01-treated cells, possibly just prior to Cdc2 activation and entry of DNA-damaged cells into mitosis. This inhibition appeared, however, to be a consequence of a further upstream action since in vitro studies revealed purified Wee1Hu was relatively resistant to UCN-01-inhibition. Consistent with such an upstream action, UCN-01 also promoted the hyperphosphorylation (activation) of Cdc25C in DNA-damaged cells. Our results suggest that UCN-01 abrogates G2 checkpoint function through inhibition of a kinase residing upstream of Cdc2, Wee1Hu, and Cdc25C, and that changes observed in these mitotic regulators are downstream consequences of UCN-01's actions.

Alkaloids↗
PubMed1999

Cellular responses to excess phospholipid.

Phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells, and its synthesis is controlled by the activity of CDP:phosphocholine cytidylyltransferase (CCT). Enforced CCT expression accelerated the rate of PtdCho synthesis. However, the amount of cellular PtdCho did not increase as a result of the turnover of both the choline and glycerol components of PtdCho. Metabolic labeling experiments demonstrated that cells compensated for elevated CCT activity by the degradation of PtdCho to glycerophosphocholine (GPC). Phospholipase D-mediated PtdCho hydrolysis and phosphocholine formation were unaffected. Most of the GPC produced in response to excess phospholipid production was secreted into the medium. Cells also degraded the excess membrane PtdCho to GPC when phospholipid formation was increased by exposure to exogenous lysophosphatidylcholine or lysophosphatidylethanolamine. The replacement of the acyl moiety at the 1-position of PtdCho with a non-hydrolyzable alkyl moiety prevented degradation to GPC. Accumulation of alkylacyl-PtdCho was associated with the inhibition of cell proliferation, demonstrating that alternative pathways of degradation will not substitute. GPC formation was blocked by bromoenol lactone, implicating the calcium-independent phospholipase A2 as a key participant in the response to excess phospholipid. Owing to the fact that PtdCho is biosynthetically converted to PtdEtn, excess PtdCho resulted in overproduction and exit of GPE as well as GPC. Thus, general membrane phospholipid homeostasis is achieved by a balance between the opposing activities of CCT and phospholipase A2.

Acylation↗
PubMed1999

Studies of the role of group VI phospholipase A2 in fatty acid incorporation, phospholipid remodeling, lysophosphatidylcholine generation, and secretagogue-induced arachidonic acid release in pancreatic islets and insulinoma cells.

An 84-kDa group VI phospholipase A2 (iPLA2) that does not require Ca2+ for catalysis has been cloned from Chinese hamster ovary cells, murine P388D1 cells, and pancreatic islet beta-cells. A housekeeping role for iPLA2 in generating lysophosphatidylcholine (LPC) acceptors for arachidonic acid incorporation into phosphatidylcholine (PC) has been proposed because iPLA2 inhibition reduces LPC levels and suppresses arachidonate incorporation and phospholipid remodeling in P388D1 cells. Because islet beta-cell phospholipids are enriched in arachidonate, we have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1 insulinoma cells. Inhibition of iPLA2 with a bromoenol lactone (BEL) suicide substrate did not suppress and generally enhanced [3H]arachidonate incorporation into these cells in the presence or absence of extracellular calcium at varied time points and BEL concentrations. Arachidonate incorporation into islet phospholipids involved deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular glucose concentrations and by examining [14C]glucose incorporation into phospholipids. BEL also inhibited islet cytosolic phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor propranolol did not affect arachidonate incorporation into islet or INS-1 cell phospholipids. Inhibition of islet iPLA2 did not alter the phospholipid head-group classes into which [3H]arachidonate was initially incorporated or its subsequent transfer from PC to other lipids. Electrospray ionization mass spectrometric measurements indicated that inhibition of INS-1 cell iPLA2 accelerated arachidonate incorporation into PC and that inhibition of islet iPLA2 reduced LPC levels by 25%, suggesting that LPC mass does not limit arachidonate incorporation into islet PC. Gas chromatography/mass spectrometry measurements indicated that BEL but not propranolol suppressed insulin secretagogue-induced hydrolysis of arachidonate from islet phospholipids. In islets and INS-1 cells, iPLA2 is thus not required for arachidonate incorporation or phospholipid remodeling and may play other roles in these cells.

Animals↗
PubMed1999

Involvement of group VI Ca2+-independent phospholipase A2 in protein kinase C-dependent arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells.

We investigated the possible involvement of group VI Ca2+-independent phospholipase A2 (iPLA2) in arachidonic acid (AA) liberation in zymosan-stimulated macrophage-like P388D1 cells. Zymosan-induced AA liberation was markedly inhibited by methyl arachidonoyl fluorophosphonate, a dual inhibitor of group IV cytosolic phospholipase A2 (cPLA2) and iPLA2. We found that a relatively specific iPLA2 inhibitor, bromoenol lactone, significantly decreased the zymosan-induced AA liberation in parallel with the decrease in iPLA2 activity, without an effect on diacylglycerol formation. Consistent with this, attenuation of iPLA2 activity by a group VI iPLA2 antisense oligonucleotide resulted in a decrease in zymosan-induced prostaglandin D2 generation. These findings suggest that zymosan-induced AA liberation may be, at least in part, mediated by iPLA2. A protein kinase C (PKC) inhibitor diminished zymosan-induced AA liberation, while a PKC activator, phorbol 12-myristate 13-acetate (PMA), enhanced the liberation. Bromoenol lactone suppressed the PMA-enhanced AA liberation without any effect on PMA-induced PKC activation. Down-regulation of PKCalpha on prolonged exposure to PMA also decreased zymosan-induced AA liberation. Under these conditions, the remaining AA liberation was insensitive to bromoenol lactone. Furthermore, the PKC depletion suppressed increases in iPLA2 proteins and the activity in the membrane fraction of zymosan-stimulated cells. In contrast, the zymosan-induced increases in iPLA2 proteins and the activity in the fraction were facilitated by simultaneous addition of PMA. Although intracellular Ca2+ depletion prevented zymosan-induced AA liberation, the translocation of PKCalpha to membranes was also inhibited. Taken together, we propose that zymosan may stimulate iPLA2-mediated AA liberation, probably through a PKC-dependent mechanism.

Animals↗
PubMed1999

Importance of the beta12-beta13 loop in protein phosphatase-1 catalytic subunit for inhibition by toxins and mammalian protein inhibitors.

Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the beta12-beta13 loop attenuated the inhibition of the resulting PP1alpha catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1alpha, the PP1alpha catalytic core, and CRHM2 with identical IC(50). Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the beta12-beta13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the beta12-beta13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.

Alkenes↗
PubMed1999

Regulation of cyclooxygenase-2 expression by phosphatidate phosphohydrolase in human amnionic WISH cells.

Prostaglandins are known to play a key role in the initiation of labor in humans, but the mechanisms governing their synthesis in amnion are largely unknown. In this study, we have examined the regulatory pathways for prostaglandin E(2) (PGE(2)) production during protein kinase C-dependent activation of human WISH cells. In these cells, PGE(2) synthesis appears to be limited not by free arachidonic acid availability but by the expression levels of cyclooxygenase-2 (COX-2). Concomitant with the cells being able to synthesize and secrete PGE(2), we detected significant elevations of both COX-2 protein and mRNA levels. Specific inhibition of COX-2 by NS-398 totally ablated PGE(2) synthesis. All of these responses were found to be strikingly dependent on an active phosphatidate phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 activity by three different strategies (i.e. use of bromoenol lactone, propranolol, and ethanol) resulted in inhibition of COX-2 expression and hence of PGE(2) production. These data unveil a novel signaling mechanism for the regulation of PGE(2) production via regulation of COX-2 expression and implicate phosphatidate phosphohydrolase 1 as a key regulatory component of eicosanoid metabolic pathways in the amnion.

Amnion↗
PubMed2000

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain.

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.

Amides↗
PubMed2000

A Ca(2+)-independent activation of a type IV cytosolic phospholipase A(2) underlies the receptor stimulation of arachidonic acid-dependent noncapacitative calcium entry.

The oscillatory [Ca(2+)](i) signals typically seen following physiologically relevant stimulation of phospholipase C-linked receptors are associated with a receptor-activated entry of Ca(2+), which plays a critical role in driving the oscillations and influencing their frequency. We have recently shown that this receptor-activated entry of Ca(2+) does not conform to the widely accepted "capacitative" model and, instead, reflects the activity of a distinct, novel Ca(2+) entry pathway regulated by arachidonic acid (Shuttleworth, T. J., and Thompson, J. L. (1998) J. Biol. Chem. 273, 32636-32643). We now show that the generation of arachidonic acid under these conditions results from the activity of a type IV cytosolic phospholipase A(2) (cPLA(2)). Although cPLA(2) activation commonly involves a Ca(2+)-dependent translocation to the membrane, at these low agonist concentrations cPLA(2) activation was independent of increases in [Ca(2+)](i), and no detectable translocation to the membrane occurs. Nevertheless, stimulation of cPLA(2) activity was confined to the membrane fraction, where an increase in phosphorylation of the enzyme was observed. We suggest that, at the low agonist concentrations associated with oscillatory [Ca(2+)](i) signals, cPLA(2) activation involves an increased phosphorylation of a discrete pool of the total cellular cPLA(2) that is already localized within the membrane fraction at resting [Ca(2+)](i).

Arachidonic Acid↗
PubMed2000

Human calcium-independent phospholipase A2 mediates lymphocyte proliferation.

Activation of lymphocytes induces blastogenesis and cell division which is accompanied by membrane lipid metabolism such as increased fatty acid turnover. To date little is known about the enzymatic mechanism(s) regulating this process. Release of fatty acids such as arachidonic acid requires sn-2-deacylation catalyzed by a class of enzymes known as phospholipases A(2) (PLA(2), EC ). Herein, we confirm that human peripheral blood B or T lymphocytes (PBL) do not possess measurable levels of 85-kDa PLA(2) as assessed by Western immunoblot. Low levels of 14-kDa PLA(2) protein and activity were detectable in the particulate fraction of PBL and Jurkat cells. Western immunoblot analysis indicates that PBLs possess the calcium-independent PLA(2) (iPLA(2)) protein. Calcium-independent sn-2-acylhydrolytic activity was measurable in PBL cytosols and could be inhibited by the selective iPLA(2) inhibitor bromoenol lactone. Mitogen activation of PBLs resulted in maintenance of activity levels which remained constant over 72 h suggesting an important role for iPLA(2) in this proliferative process. Indeed, evaluation of iPLA(2) activity in cell cycle-arrested Jurkat T cell fractions revealed the highest iPLA(2) levels occurring at the G(2)/M phase. Addition of the iPLA(2) inhibitors, bromoenol lactone, or arachidonyl trifluoromethyl ketone (AAOCF(3)), inhibited both mitogen-induced PBL as well as Jurkat T cell proliferation. Moreover, specific depletion of iPLA(2) protein by antisense treatment also resulted in marked suppression of cell division. Inhibition of Jurkat cell proliferation was not associated with arrest at a particular phase of the cell cycle nor was it associated with apoptosis as assessed by flow cytometry. These findings provide the first evidence that iPLA(2) plays a key role in the lymphocyte proliferative response.

Apoptosis↗
PubMed2001

Anti-CD3 and concanavalin A-induced human T cell proliferation is associated with an increased rate of arachidonate-phospholipid remodeling. Lack of involvement of group IV and group VI phospholipase A2 in remodeling and increased susceptibility of proliferating T cells to CoA-independent transacyclase inhibitor-induced apoptosis.

In this study arachidonate-phospholipid remodeling was investigated in resting and proliferating human T lymphocytes. Lymphocytes induced to proliferate with either the mitogen concanavalin A or with anti-CD3 (OKT3) in combination with interleukin 2 (OKT3/IL-2) showed a greatly accelerated rate of [3H]arachidonate-phospholipid remodeling compared with resting lymphocytes or with lymphocytes stimulated with OKT3 or IL-2 alone. The concanavalin A-stimulated cells showed a 2-fold increase in the specific activity of CoA-independent transacylase compared with unstimulated cells, indicating that this enzyme is inducible. Stimulation with OKT3 resulted in greatly increased quantities of the group VI calcium-independent phospholipase A2 but not of the quantity of group IV cytosolic phospholipase A2. However, group IV phospholipase A2 became phosphorylated in OKT3-stimulated cells, as determined by decreased electrophoretic mobility. Incubation of cells with the group VI phospholipase A2 inhibitor, bromoenol lactone, or the dual group IV/group VI phospholipase A2 inhibitor, methyl arachidonyl fluorophosphonate, did not block arachidonate-phospholipid remodeling resting or proliferating T cells, suggesting that these phospholipases A2 were not involved in arachidonate-phospholipid remodeling. The incubation of nonproliferating human lymphocytes with inhibitors of CoA-independent transacylase had little impact on cell survival. In contrast, OKT3/IL-2-stimulated T lymphocytes were very sensitive to apoptosis induced by CoA-independent transacylase inhibitors. Altogether these results indicate that increased arachidonate-phospholipid remodeling is associated with T cell proliferation and that CoA-independent transacylase may be a novel therapeutic target for proliferative disorders.

Acyltransferases↗
PubMed2001

The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gqalpha -dependent ERK-independent pathway.

The Ca(2+)-sensing receptor (CaR) stimulates a number of phospholipase activities, but the specific phospholipases and the mechanisms by which the CaR activates them are not defined. We investigated regulation of phospholipase A(2) (PLA(2)) by the Ca(2+)-sensing receptor (CaR) in human embryonic kidney 293 cells that express either the wild-type receptor or a nonfunctional mutant (R796W) CaR. The PLA(2) activity was attributable to cytosolic PLA(2) (cPLA(2)) based on its inhibition by arachidonyl trifluoromethyl ketone, lack of inhibition by bromoenol lactone, and enhancement of the CaR-stimulated phospholipase activity by coexpression of a cDNA encoding the 85-kDa human cPLA(2). No CaR-stimulated cPLA(2) activity was found in the cells that expressed the mutant CaR. Pertussis toxin treatment had a minimal effect on CaR-stimulated arachidonic acid release and the CaR-stimulated rise in intracellular Ca(2+) (Ca(2+)(i)), whereas inhibition of phospholipase C (PLC) with completely inhibited CaR-stimulated PLC and cPLA(2) activities. CaR-stimulated PLC activity was inhibited by expression of RGS4, an RGS (Regulator of G protein Signaling) protein that inhibits Galpha(q) activity. CaR-stimulated cPLA(2) activity was inhibited 80% by chelation of extracellular Ca(2+) and depletion of intracellular Ca(2+) with EGTA and inhibited 90% by treatment with W7, a calmodulin inhibitor, or with KN-93, an inhibitor of Ca(2+), calmodulin-dependent protein kinases. Chemical inhibitors of the ERK activator, MEK, and a dominant negative MEK, MEK(K97R), had no effect on CaR-stimulated cPLA(2) activity but inhibited CaR-stimulated ERK activity. These results demonstrate that the CaR activates cPLA(2) via a Galpha(q), PLC, Ca(2+)-CaM, and calmodulin-dependent protein kinase-dependent pathway that is independent the ERK pathway.

Arachidonic Acid↗
PubMed2001

Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta.

A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Protein phosphatases regulate DNA-dependent protein kinase activity.

DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA end-binding Ku70/Ku80 heterodimer. DNA-PK is required for DNA double strand break repair by the process of nonhomologous end joining. Nonhomologous end joining is a major mechanism for the repair of DNA double strand breaks in mammalian cells. As such, DNA-PK plays essential roles in the cellular response to ionizing radiation and in V(D)J recombination. In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku70 and Ku80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK. Here we show that phosphorylation-induced loss of the protein kinase activity of DNA-PK is restored by the addition of the purified catalytic subunit of either protein phosphatase 1 or protein phosphatase 2A (PP2A) and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin. We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, causes a 50-60% decrease in DNA-PK protein kinase activity, although the protein phosphatase 1 activity in these cells was unaffected. In vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was observed when cells were labeled with [(32)P]inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid. Together, our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity and that the protein phosphatase responsible for reactivation in vivo is a PP2A-like enzyme.

Alkenes↗
PubMed2001

A plant phytotoxin, solanapyrone A, is an inhibitor of DNA polymerase beta and lambda.

Solanapyrone A, a phytotoxin and enzyme inhibitor isolated from a fungus (SUT 01B1-2) selectively inhibits the activities of mammalian DNA polymerase beta and lambda (pol beta and lambda) in vitro. The IC50 values of the compound were 30 microm for pol beta and 37 microm for pol lambda. Because pol beta and lambda are in a family and their three-dimensional structures are thought to be highly similar to each other, we used pol beta to analyze the biochemical relationship with solanapyrone A. On pol beta, solanapyrone A antagonistically competed with both the DNA template and the nucleotide substrate. BIAcore analysis demonstrated that solanapyrone A bound selectively to the N-terminal 8-kDa domain of pol beta. This domain is known to bind single-stranded DNA, provide 5'-phosphate recognition of gapped DNA, and cleave the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity) including 5'-deoxyribose phosphate lyase activity. Solanapyrone A inhibited the single-stranded DNA-binding activity but did not influence the activities of the 5'-phosphate recognition in gapped DNA structures and the AP lyase. Based on these results, the inhibitory mechanism of solanapyrone A is discussed.

DNA↗
PubMed2002

Nerve growth factor-induced p75-mediated death of cultured hippocampal neurons is age-dependent and transduced through ceramide generated by neutral sphingomyelinase.

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75(exonIII-/-) mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase(-/-) mice were as susceptible to NGF-induced death as wild type neurons. The acid ceramidase inhibitor, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, enhanced cell death, supporting a role for ceramide itself and not a downstream lipid metabolite. Finally, scyphostatin inhibited NGF-induced Jnk phosphorylation in hippocampal neurons. These data indicate an initiating role of ceramide generated by neutral sphingomyelinase in the diverse neuronal responses induced by binding of neurotrophins to p75.

Age Factors↗
PubMed2002

Oxyresveratrol and hydroxystilbene compounds. Inhibitory effect on tyrosinase and mechanism of action.

Tyrosinase is responsible for the molting process in insects, undesirable browning of fruits and vegetables, and coloring of skin, hair, and eyes in animals. To clarify the mechanism of the depigmenting property of hydroxystilbene compounds, inhibitory actions of oxyresveratrol and its analogs on tyrosinases from mushroom and murine melanoma B-16 have been elucidated in this study. Oxyresveratrol showed potent inhibitory effect with an IC(50) value of 1.2 microm on mushroom tyrosinase activity, which was 32-fold stronger inhibition than kojic acid, a depigmenting agent used as the cosmetic material with skin-whitening effect and the medical agent for hyperpigmentation disorders. Hydroxystilbene compounds of resveratrol, 3,5-dihydroxy-4'-methoxystilbene, and rhapontigenin also showed more than 50% inhibition at 100 microm on mushroom tyrosinase activity, but other methylated or glycosylated hydroxystilbenes of 3,4'-dimethoxy-5-hydroxystilbene, trimethylresveratrol, piceid, and rhaponticin did not inhibit significantly. None of the hydroxystilbene compounds except oxyresveratrol exhibited more than 50% inhibition at 100 microm on l-tyrosine oxidation by murine tyrosinase activity; oxyresveratrol showed an IC(50) value of 52.7 microm on the enzyme activity. The kinetics and mechanism for inhibition of mushroom tyrosinase exhibited the reversibility of oxyresveratrol as a noncompetitive inhibitor with l-tyrosine as the substrate. The interaction between oxyresveratrol and tyrosinase exhibited a high affinity reflected in a K(i) value of 3.2-4.2 x 10(-7) m. Oxyresveratrol did not affect the promoter activity of the tyrosinase gene in murine melanoma B-16 at 10 and 100 microm. Therefore, the depigmenting effect of oxyresveratrol works through reversible inhibition of tyrosinase activity rather than suppression of the expression and synthesis of the enzyme. The number and position of hydroxy substituents seem to play an important role in the inhibitory effects of hydroxystilbene compounds on tyrosinase activity.

Agaricales↗