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Phosphatidylcholine (PC) is the main constituent of mammalian cell membranes. Consequently, preservation of membrane PC content and composition - PC homeostasis - is crucial to maintain cellular life. PC biosynthetic pathway is generally controlled by CTP:phosphocholine cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCTα is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum redistribution. However, most of the enzyme is located inside the nuclei. Here, we demonstrate that CCTα is the most abundant isoform in renal collecting duct cells, and its redistribution is dependent on endogenous prostaglandins. Previously we have demonstrated that PC synthesis was inhibited by indomethacin (Indo) treatment, and this effect was reverted by exogenous PGD(2). In this work we found that Indo induced CCTα distribution into intranuclear Lamin A/C foci. Exogenous PGD(2) reverted this effect by inducing CCTα redistribution to nuclear envelope, suggesting that PGD(2) maintains PC synthesis by CCTα mobilization. Interestingly, we found that the effect of PGD(2) was dependent on ERK1/2 activation. In conclusion, our previous observations and the present results lead us to suggest that papillary cells possess the ability to maintain their structural integrity through the synthesis of their own survival molecule, PGD(2), by modulating CCTα intracellular location., (Copyright © 2016. Published by Elsevier B.V.)

Accumulating evidence has suggested that cytosolic phospholipase A(2) (cPLA(2)) and several secretory PLA(2) (sPLA(2)) isozymes are signaling PLA(2)s that are functionally coupled with downstream cyclooxygenase (COX) isozymes for prostaglandin (PG) biosynthesis. Arachidonic acid (AA) released by cPLA(2) and sPLA(2)s is supplied to both COX-1 and COX-2 in the immediate, and predominantly to COX-2 in the delayed, PG-biosynthetic responses. Vimentin, an intermediate filament component, acts as a functional perinuclear adapter for cPLA(2), in which the C2 domain of cPLA(2) associates with the head domain of vimentin in a Ca(2+)-sensitive manner. The heparin-binding signaling sPLA(2)-IIA, IID and V bind the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican, which plays a role in sorting of these isozymes into caveolae and perinuclear compartments. Phospholipid scramblase, which facilitates transbilayer movement of anionic phospholipids, renders the cellular membranes more susceptible to signaling sPLA(2)s. There is functional cooperation between cPLA(2) and signaling sPLA(2)s in that prior activation of cPLA(2) is required for the signaling sPLA(2)s to act properly. cPLA(2)-derived AA is oxidized by 12/15-lipoxygenase, the products of which not only augment the induction of sPLA(2) expression, but also cause membrane perturbation, leading to increased cellular susceptibility to the signaling sPLA(2)s. sPLA(2)-X, a heparin-non-binding sPLA(2) isozyme, is capable of releasing AA from intact cells in the absence of cofactors. This property is attributed to its ability to avidly hydrolyze zwitterionic phosphatidylcholine, a major phospholipid in the outer plasma membrane. sPLA(2)-V can also utilize this route in several cell types. Taken together, the AA-releasing function of sPLA(2)s depends on the presence of regulatory cofactors and interfacial binding to membrane phospholipids, which differ according to cell type, stimuli, secretory processes, and subcellular distributions.

Liberation of arachidonic acid by cytosolic phospholipase A(2) (cPLA(2)) upon cell activation is often the initial and rate-limiting step in leukotriene and prostaglandin biosynthesis. This review discusses the essential features of cPLA(2) isoforms and addresses intriguing insights into the catalytic and regulatory mechanisms. Gene expression, posttranslational modification and subcellular localization can regulate these isoforms. Translocation of cPLA(2)alpha from the cytosol to the perinuclear region in response to calcium transients is critical for the immediate arachidonic acid release. Therefore, particular emphasis is placed on the mechanism of the translocation and the role of the proteins and lipids implicated in this process. The regional distribution and cellular localization of cPLA(2) may help to better understand its function as an arachidonic acid supplier to downstream enzymes and as a regulator of specific cellular processes.

We previously reported that unsaturated long-chain fatty acids were potent DNA polymerase inhibitors (Y. Mizushina et al., J. Biol. Chem. 274 (1999) 25599-25607). In those experiments, the question remained of whether metastable oil droplets (liposomal vesicles) of the unsaturated long-chain fatty acids can non-specifically inhibit the polymerase activity. We report here that only the soluble fatty acid monomers of linoleic acid or nervonic acid could affect the activities of mammalian DNA polymerases, and the metastable oil droplets could not. When we consider the facts that nuclear membranes are a kind of liposomal vesicles, that free fatty acids occur only at the moment the lipids are digested, and that the DNA polymerization possibly occurs on the nuclear membranes, the data shown here are suggestive regarding the mechanism of regulation of DNA polymerization in vivo.

Lysophospholipases play essential roles in keeping their multi-functional substrates, the lysophospholipids, at safe levels. Recently, a 25 kDa human lysophospholipase A (hLysoPLA I) that is highly conserved among rat, mouse, human and rabbit has been cloned, expressed and characterized and appears to hydrolyze only lysophospholipids among the various lipid substrates. Interestingly, this enzyme also displays acyl-protein thioesterase activity towards a G protein alpha subunit. To target the subcellular location of this hLysoPLA I, we have carried out immunocytochemical studies and report here that hLysoPLA I appears to be associated with the endoplasmic reticulum (ER) and nuclear envelope in human amnionic WISH cells and not the plasma membrane. In addition, we found that the hLysoPLA I can be up-regulated by phorbol 12-myristate 13-acetate (PMA) stimulation, a process in which phospholipase A(2) is activated and lysophospholipids are generated in WISH cells. Furthermore, the PMA-induced hLysoPLA I expression can be blocked by the protein kinase C (PKC) inhibitor Gö6976. The regulated expression of the LysoPLA/acyl-protein thioesterase by PKC may have important implications for signal transduction processes.

To obtain more insight into the mechanisms regulating CTP:phosphocholine cytidylyltransferase (CT), we determined the effect of oleate treatment on the rate of CT release from permeabilized Hep G2 cells and the distribution of the CT remaining in the permeabilized cells. When we permeabilized untreated cells in pH 7.5 buffer containing 0.15 M KCl, the rate of CT release was much slower than the release of lactate dehydrogenase. Oleate treatment caused a further decrease in CT release from cells. In untreated cells, 70-80% of the CT remaining in cells 10 min after permeabilization was recovered as soluble CT. Oleate treatment increased the amount of bound CT but over 50% of the CT in cells 10 min after permeabilization was recovered as soluble CT. In both control and oleate-treated cells, the increase in CT release with time correlated with a decrease in the amount of CT recovered from permeabilized cells as soluble CT. These results suggested that CT existed in a form that was not immediately available for release from permeabilized cells, but was recovered in the soluble fraction after cell disruption. When cells were permeabilized in 10 mM imidazole-20% glycerol-5 mM Mg2+ pH 6.5, over 80% of CT in control and over 90% of CT in oleate-treated cells was recovered bound to the particulate fraction. Essentially no CT was released from the cells. The recovery of CT in the particulate fraction required Mg2+ to be present when permeabilization was initiated. The addition of Mg2+, after cells were disrupted, did not increase CT in the particulate fraction. In untreated cells, 50% of bound CT was active. Oleate treatment increased the amount of active CT in the particulate fraction to over 70% of total. About 50% of particulate CT in untreated cells but only 15% in oleate-treated cells was extracted with 0.15 M KCl. Inactive CT was preferentially extracted by KCl. The bound CT was recovered in isolated nuclei. Overall, the results suggested that both inactive and active CT are bound to nuclear membranes, and that the activation of CT involves conversion of CT loosely bound to membrane to a form more tightly bound to membranes perhaps by hydrophobic interaction with phospholipids. This model does not involve translocation from a soluble pool., (Copyright 1998 Elsevier Science B.V.)

Neuronal nuclear fraction N1 was isolated from cerebral cortices of 15-day-old rabbits, and nuclear subfractions prepared, in order to study the location of nuclear lyso platelet-activating factor (lyso-PAF) acetyltransferase and alkylglycerophosphate (AGP) acetyltransferase, and factors that affect the loss of these two nuclear activities. Subfractionation of prelabelled N1 indicated that the nuclear envelope had the highest percentage of the radioactive acetylated products alkylacetylglycerophosphate (AAGP) and PAF, and the distribution of these phospholipids reflected phospholipid distributions in the nuclear subfractions. The majority (95%) of radioactive AAGP and PAF was also recovered in Triton X-100 extracts of prelabelled nuclei, suggesting that these acetylated lipids are located in nuclear membranes rather than in the nuclear matrix/chromatin. Of the nuclear subfractions, the envelope had the highest AGP and lyso-PAF acetyltransferase specific activities which were close to corresponding values seen in the parent N1 fraction. Thus the nuclear AGP and lyso-PAF acetyltransferases were principally localized to the nuclear membranes. Differentials in activity loss were seen for the two acetyltransferase activities. In the nuclear envelope fractions, the lyso-PAF acetyltransferase was the more susceptible to oxidation reactions which could be reversed or blocked by the use of reducing agents. In preincubations, N1 showed greater losses in lyso-PAF acetyltransferase activity than in AGP acetyltransferase activity, losses which were not attributable to oxidation. Addition of cytosolic fraction S3 to preincubations promoted losses for each acetyltransferase in N1, and gave evidence for cytosolic and endogenous nuclear contributions to the activity loss. Addition of okadaic acid to the preincubations did not prevent the decline of either acetyltransferase in intact nuclei, but did diminish the loss of nuclear lyso-PAF acetyltransferase activity promoted by S3 addition, and also blocked the loss of this acetyltransferase seen in preincubations of isolated nuclear envelopes. This suggests that nuclear lyso-PAF acetyltransferase is susceptible to okadaic acid-sensitive nuclear and cytosolic protein phosphatase activities, while AGP acetyltransferase may lose activity by the action of other phosphatases or by other mechanisms within the nucleus.

A 46 kDa ATP binding polypeptide of the nuclear envelope, virtually identical to the nuclear envelope NTPase putatively involved in mRNA efflux [6], is present in all rat liver cell membranes. Its presence in nuclear envelope is not the result of cross contamination during isolation.

The activities of nuclear envelope-associated protein phosphokinase and protein phosphatase were determined in nuclear ghosts from liver and oviduct of quails. The protein kinase was found to be inhibited by poly(A) by 75%. During the kinase reaction proteins with molecular weights of 106 000 and 64 000 were phosphorylated. The phosphoprotein phosphatase from liver was stimulated to 190% by poly(A), whereas only a slight enhancing effect by this polymer was determined with the oviduct enzyme (to 125%). Comparative determinations of the nuclear ghost-associated enzyme activities revealed the following values (in nmol Pi/min per 10(8) ghosts); oviduct: phosphokinase, 0.015; phosphatase, 0.004 and nucleoside triphosphatase, 39.4; and liver: phosphokinase, 0.044; phosphatase, 0.012 and nucleoside triphosphatase, 11.7. These data indicate that phosphorylation/dephosphorylation proceeds independently of the nucleoside triphosphatase cycle. This assumption is supported by analytical results revealing that no marked dephosphorylation occurs after poly(A) binding to the nuclear envelope. Moreover, stoichiometrical data showed a nearly 1:1 molar ratio between ATP-binding and phosphorylation of nuclear envelope protein. From these findings a new model for the nucleoside triphosphatase-mediated poly(A)(+)mRNA efflux from nuclei is deducted, proposing phosphokinase and phosphatase only to modulate the affinity of the 'carrier structure' for poly(A) (+)mRNA, but not to constitute the nucleoside triphosphatase.

Nucleocytoplasmic transport of rat liver mRNA is thought to be regulated by a nucleoside triphosphatase whose activity in the intact nuclear envelope is stimulated by the 3'poly(A) tail of poly(A)+ mRNA. In contrast to the liver mRNA, the mRNA from rat brain contains a great population of poly(A)- mRNA's that does not appear until after birth. Measurements of the nuclear-envelope-associated enzyme activities involved in mRNA transport, and their dependence on endogenous (isolated cytoplasmic mRNA-transport-stimulating proteins) and exogenous (poly(A), lectins, and neoglycoproteins) factors during prenatal and postnatal rat brain and liver development, revealed marked organ-dependent differences paralleling the appearance of the poly(A)- mRNA unique in the brain.

We have isolated a nuclear membrane fraction from KB cells infected with human adenovirus 2 that synthesizes exclusively small viral DNA chains (approx. 9 S) in vitro (Yamashita, T., Arens, M. and Green, M. (1975) J. Biol. Chem. 250, 3273-3279). The DNA polymerase activity present in the adenovirus 2 DNA-nuclear membrane complex was purified through chromatography on phosphocellulose and DEAE-cellulose, glycerol gradient centrifuation and DNA-cellulose chromatography. A single peak of enzymatic activity sedimented in glycerol gradients at about 6.7 S which corresponds to a molecular weight of 125000. The enzyme preparation in the step of glycerol gradient centrifugation utilized activated calf thymus, KB cell and adenovirus 2 DNA as template-primer in the presence of Mg2+; Km values for these DNAs were 5.5, 4.0, and 0.8 mug/ml, respectively. The partially purified enzyme preparation was characterized by several criteria which were compared to the properties of the three major mammalian DNA polymerases, alpha, beta, and psi. On the basis of template-primer preference, effect of salt, inhibition by N-ethylmaleimide and Km for dTTP, the DNA polymerase activity from the membrane complex can be distinguished from the alpha and beta DNA polymerases. The elution profile from DNA cellulose revealed a minor peak (I) and a major peak (II) of DNA polymerase activity utilizing poly(A) -(dT)10 as template-primer in the presence of Mn2+ - Peak II from DNA cellulose, which contained about 90% of the total DNA polymerase activity eluted from the column, was 2-3 times as active with poly(A) - (dT)10 as template-primer in the presence of Mn2+ than with activated calf thymus DNA in the presence of Mg2+. On the other hand, peak I had a low ratio of poly(A) - (dT)10 to activated calf thymus DNA activity. DNA polymerase was also purified from the nuclear membrane fraction of uninfected KB cells by the same procedures as those used in enzyme purification from the adenovirus 2 DNA-nuclear membrane complex. A minor peak and a major peak of DNA polymerase activity utilizing poly(A) - (dT)10 as template primer in the presence of Mn2+ were again observed that eluted from DNA cellulose at the same KCl concentrations as peak I and II from adenovirus 2-infected cells. The enzymes of the nuclear membrane fraction of uninfected KB cells could not be differentiated from the enzymes of the adenovirus 2 DNA-nuclear membrane complex through any of the purification steps nor by their template specificities. These results indicate that the predominant enzyme in the adenovirus 2 DNA-nuclear membrane complex and in the KB cell nuclear membrane complex belongs to the class of DNA polymerase psi.

Nuclei were prepared from monkey hepatocytes by centrifugation of the homogenate on a cushion of 2.3 M sucrose, during 45 min at 100000 X g. The yield was 2.2 x 10(7) nuclei per g of liver, and 70% of te homogenate DNA was recovered in these nuclei. An electron microscopic study as well as a biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. A mannosyltransferase and an N-acetylglucosaminyltransferase, working on endogenous glycoproteic acceptors, are present in the nuclei for 1.4 and 6.5% of the homogenate activities, respectively. The nuclei are hydrolysed by DNAse I. The suspension, adjusted in 1.9 M sucrose, was centrifuged for 2 h at 100000 X g, under buffer layer. Purified nuclear membranes were collected at the interface. These membranes did not contain any more endoplasmic reticulum enzyme activities, but the mannosyl and N-acetylglucosaminyltransferase activities were still present. They essentially work on an exogenous chromatin acceptor, prepared by lysis of the nuclei. The eventual role of these glycosyltransferases in the glycosylation of non-histone proteins is discussed.

delta 4-Steroid-5 alpha-reductase (3-oxo-5 alpha-steroid:NADP+ delta 4-oxidoreductase, EC 1.3.1.22), is a membrane-bound enzyme. In the ventral prostate of the rat, its activity is found within the nuclear envelope. Solubilization of this enzyme can only be achieved in the presence of detergents. We studied the inhibitory effect of various detergents on 5 alpha-reductase activity as a function of detergent concentration, of pH, of incubation time, of salt concentration and of additives to the buffer system. Four detergents (Lubrol WX, CHAPS, L-alpha-lysophosphatidylcholine and octyl D-glucopyranoside) were selected for subsequent solubilization studies. The overall recovery of solubilized enzyme activity was about 30% when compared to 100% of 5 alpha-reductase activity found in freshly prepared nuclei. Up to 20-30% of the nuclear proteins were extracted during the solubilization procedure. Among the various treatments tested, a concentration of 3 mg/ml L-alpha-lysophosphatidylcholine per 10 mg/ml of nuclear protein in the presence of 5 mM MgCl2, 0.1 M KCl, 0.1 M sodium citrate and 5 mM NADPH yielded the maximal enzymic activity of 56%, 15% of the nuclear proteins being solubilized in an active and stable form. The activity in these extracts could be kept stable for 2 days at 4 degrees C with a recovery of 75% of enzymic activity. A 3-fold increase of specific 5 alpha-reductase activity was obtained during solubilization under optimal conditions.