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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.)

Mutations in the nuclear envelope protein lamin A or in its processing protease ZMPSTE24 cause human accelerated aging syndromes, including Hutchinson-Gilford progeria syndrome. Similarly, Zmpste24-deficient mice accumulate unprocessed prelamin A and develop multiple progeroid symptoms, thus representing a valuable animal model for the study of these syndromes. Zmpste24-deficient mice also show marked transcriptional alterations associated with chromatin disorganization, but the molecular links between both processes are unknown. We report herein that Zmpste24-deficient mice show a hypermethylation of rDNA that reduces the transcription of ribosomal genes, being this reduction reversible upon treatment with DNA methyltransferase inhibitors. This alteration has been previously described during physiological aging in rodents, suggesting its potential role in the development of the progeroid phenotypes. We also show that Zmpste24-deficient mice present global hypoacetylation of histones H2B and H4. By using a combination of RNA sequencing and chromatin immunoprecipitation assays, we demonstrate that these histone modifications are associated with changes in the expression of several genes involved in the control of cell proliferation and metabolic processes, which may contribute to the plethora of progeroid symptoms exhibited by Zmpste24-deficient mice. The identification of these altered genes may help to clarify the molecular mechanisms underlying aging and progeroid syndromes as well as to define new targets for the treatment of these dramatic diseases., (© 2010 The Authors. Aging Cell © 2010 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Akt is involved in the regulation of diverse cellular functions such as cell proliferation, energy metabolism, and apoptosis. Although three Akt isoforms are known, the function of each isoform is poorly understood. To gain a better understanding of each Akt isoform, we examined the subcellular localization and expression of each isoform in transformed and nontransformed cells. Akt1 was localized in the cytoplasm, which is in agreement with the currently accepted model that cytoplasmic Akt is translocated and activated at the inner leaflet of the plasma membrane. Interestingly, HEK-293 and HEK-293T cells contained Akt1 in the nucleus and cytoplasm, respectively, suggesting that SV40 T-antigen plays a crucial role in the cytoplasmic localization and activation of Akt1 in HEK-293T. Akt2 was colocalized with the mitochondria, while Akt3 was localized in both the nucleus and nuclear membrane. The subcellular localization of the Akt isoforms was not substantially altered in response to ionizing radiation or EGF. Furthermore, the ablation of one Akt isoform by small interfering RNA (siRNA) did not alter the subcellular location of the remaining isoforms, suggesting that the major function of one isoform is not compensated for by other isoforms. Together, our data support the notion that Akt2 and Akt3 are regulated at the mitochondrial and nuclear membranes, respectively. The mitochondrial localization of Akt2 raises the possibility that this isoform may be involved in both glucose-based energy metabolism and suppression of apoptosis, two Akt functions previously identified with anti-pan-Akt antibodies.

The link between nucleocytoplasmic transport and apoptosis remains controversial. Nucleocytoplasmic exchange of molecules seems indeed essential for the initiation and execution of the apoptotic programme; but inhibition of nuclear transport factors may also represent a powerful apoptotic trigger. The GTPase Ran (together with its partners), first discovered to be essential in nucleocytoplasmic transport, has multiple key functions in cell biology, and particularly in spindle assembly, kinetochore function and nuclear envelope assembly. Among the Ran partners studied, NTF2 appears to be solely involved in nucleocytoplasmic transport. Here, we localised Ran and several of its partners, RanBP1, CAS and NTF2, at the nuclear membrane in the trypanosomatid Leishmania major. Remarkably, these proteins fused to GFP decorated a perinuclear 'collar' of about 15 dots, colocalising at nuclear pore complexes with the homologue of nucleoporin Sec13. In the other trypanosomatid Trypanosoma brucei, RNAi knockdown of the expression of the corresponding genes resulted in an apoptosis-like phenomenon. These phenotypes show that Ran and its partners have a key function in trypanosomatids like they have in mammals. Our data, notably those about TbNTF2 RNAi, support the idea that active nucleocytoplasmic transport is not essential for the initiation and execution of apoptosis, and, rather, the impairment of this transport constitutes an intrinsic signal for triggering PCD.

Nuclear lipid metabolism is involved in the regulation of cell proliferation. Modulation of the expression and activity of nuclear PI-phospholipase C (PI-PLC) has been reported during liver regeneration after partial hepatectomy, although it has not been determined whether different PLC isoforms play specific roles in the regulation of cell cycle progression. Here, we report evidence that the increased activity of nuclear PLCs in regenerating rat liver occurs before the peak of DNA replication and involves the enzyme activity associated to the chromatin and not that associated to the nuclear membrane. Immunocytochemical analyses indicate that PI-PLC beta(1) isoform is exclusively localized at the chromatin level, PI-PLC beta(1) co-localizes with DNA replication sites much more than PI-PLC gamma(1), which is also present at the nuclear envelope. These findings and the increased amount of PI-PLC gamma(1) occurring after the peak of DNA replication suggest that PI-PLC beta(1) and gamma(1) play different roles in cell cycle progression during regenerating liver. The increased activity of PI-PLC beta(1) constitutively present within the hepatocyte nucleus, should trigger DNA replication, whereas PI-PLC gamma(1) should be involved in G2/M phase transition through lamin phosphorylation., (Copyright 2003 Wiley-Liss, Inc.)

Signaling proteins are thought to be tightly regulated spatially and temporally in order to generate specific and localized effects. For Rac and other small guanosine triphosphatases, binding to guanosine triphosphate leads to interaction with downstream targets and regulates subcellular localization. A method called FLAIR (fluorescence activation indicator for Rho proteins) was developed to quantify the spatio-temporal dynamics of the Rac1 nucleotide state in living cells. FLAIR revealed precise spatial control of growth factor-induced Rac activation, in membrane ruffles and in a gradient of activation at the leading edge of motile cells. FLAIR exemplifies a generally applicable approach for examining spatio-temporal control of protein activity.

CD38 is a type II transmembrane glycoprotein found on both hematopoietic and non-hematopoietic cells. It is known for its involvement in the metabolism of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate, two nucleotides with calcium mobilizing activity independent of inositol trisphosphate. It is generally believed that CD38 is an integral protein with ectoenzymatic activities found mainly on the plasma membrane. Here we show that enzymatically active CD38 is present intracellularly on the nuclear envelope of rat hepatocytes. CD38 isolated from rat liver nuclei possessed both ADP-ribosyl cyclase and NADase activity. Immunofluorescence studies on rat liver cryosections and isolated nuclei localized CD38 to the nuclear envelope of hepatocytes. Subcellular localization via immunoelectron microscopy showed that CD38 is located on the inner nuclear envelope. The isolated nuclei sequestered calcium in an ATP-dependent manner. cADPR elicited a rapid calcium release from the loaded nuclei, which was independent of inositol trisphosphate and was inhibited by 8-amino-cADPR, a specific antagonist of cADPR, and ryanodine. However, nicotinic acid adenine dinucleotide phosphate failed to elicit any calcium release from the nuclear calcium stores. The nuclear localization of CD38 shown in this study suggests a novel role of CD38 in intracellular calcium signaling for non-hematopoietic cells.

We have determined that prostaglandin H synthase-2 localises strongly to the nuclear membrane as well as being found in the endoplasmic reticulum in human amnion-derived WISH cells which have been stimulated with interleukin 1beta and phorbol ester. This is consistent with findings in cells of non-reproductive origin. There is strong evidence that prostaglandin J2 derivatives, which in other tissues exhibit tumour suppressing, antiproliferative and/or differentiation promoting activities, act through binding of intracellular receptors which then enter the nucleus. In addition, some arachidonic acid derivatives are clearly generated by enzymes at the nuclear envelope and localise to sites in nuclei or bind sites in nuclei. The WISH cell line will make an excellent system for studying these perinuclear intracellular prostanoid signaling mechanisms.

We have previously shown that the nuclear membrane fluidity of rat liver, measured by fluorescence anisotropy of two probes, is higher in the hydrophobic core, with respect to the bilayer surface, in newborn rats compared to adult rats. The aim of the present research is to investigate whether the nuclear membrane fluidity influences RNA nucleocytoplasmic transport. To this end two experimental models were used: the fluidity of nuclear membrane isolated from adult rats was increased by a choline base exchange reaction, which is known to be accompanied by an increase of phosphatidylcholine unsaturated fatty acids, whereas that of nuclear membrane isolated from newborn rats was decreased by incubation with dimyristoylphosphatidylcholine-cholesterol liposomes. The RNA efflux, evaluated by using [3H]uridine, significantly increased in the adult nuclear membrane submitted to choline base exchange reaction, whereas a strong decrease in the newborn nuclear membrane enriched with cholesterol was found. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme which is correlated with mRNA transport, showed parallel variations. Therefore, for the first time, we have provided evidence that the nuclear membrane fluidity plays a regulatory role in RNA nucleocytoplasmic transport, although the mechanism by which this effect takes place remains to be clarified., (Copyright 1999 Academic Press.)

The arsenite-stimulated human ATPase (hASNA-I) protein is a distinct human ATPase whose cDNA was cloned by sequence homology to the Escherichia coli ATPase arsA. Its subcellular localization in human malignant melanoma T289 cells was examined to gain insight into the role of hASNA-I in the physiology of human cells. Immunocytochemical staining using the specific anti-hASNA-I monoclonal antibody 5G8 showed a cytoplasmic, perinuclear, and nucleolar distribution. Subcellular fractionation indicated that the cytoplasmic hASNA-I was soluble and that the perinuclear distribution was due to association with the nuclear membrane rather than with the endoplasmic reticulum. Its presence in the nucleolus was confirmed by showing colocalization with an antibody of known nucleolar specificity. Further immunocytochemical analysis showed that the hASNA-I at the nuclear membrane was associated with invaginations into the nucleus in interphase cells. These results indicate that hASNA-I is a paralogue of the bacterial ArsA protein and suggest that it plays a role in the nucleocytoplasmic transport of a nucleolar component.

Eukaryotes respond to the presence of unfolded protein in the endoplasmic reticulum (ER) by up-regulating the transcription of genes encoding ER protein chaperones, such as BiP. We have isolated a novel human cDNA encoding a homolog to Saccharomyces cerevisiae Ire1p, a proximal sensor for this signal transduction pathway in yeast. The gene product hIre1p is a type 1 transmembrane protein containing a cytoplasmic domain that is highly conserved to the yeast counterpart having a Ser/Thr protein kinase domain and a domain homologous to RNase L. However, the luminal domain has extensively diverged from the yeast gene product. hIre1p expressed in mammalian cells displayed intrinsic autophosphorylation activity and an endoribonuclease activity that cleaved the 5' splice site of yeast HAC1 mRNA, a substrate for the endoribonuclease activity of yeast Ire1p. Overexpressed hIre1p was localized to the ER with particular concentration around the nuclear envelope and some colocalization with the nuclear pore complex. Expression of Ire1p mRNA was autoregulated through a process that required a functional hIre1p kinase activity. Finally, overexpression of wild-type hIre1p constitutively activated a reporter gene under transcriptional control of the rat BiP promoter, whereas expression of a catalytically inactive hIre1p acted in a trans-dominant-negative manner to prevent transcriptional activation of the BiP promoter in response to ER stress induced by inhibition of N-linked glycosylation. These results demonstrate that hIre1p is an essential proximal sensor of the unfolded protein response pathway in mammalian cells.

The possibility that interaction of the nuclear localization signal (NLS) with its pore receptor may directly stimulate nuclear envelope-associated ATPase activity and consequently provide energy for protein translocation across the pore has been studied. ATPase activity was assayed after cross-linking of the prototype NLS peptide with its pore receptor, or after preincubation of envelopes with NLS-albumin conjugates. Neither treatment enhanced enzyme activity. A more complex series of events may be required for energy-generation at the nuclear pore.

The protein kinase C (PKC) alpha, beta and epsilon isoforms have distinct nuclear localizations in neuroblastoma x glioma hybrid cells NG 108-15. We found by immunoblotting that PKC alpha, beta II, delta and epsilon are the predominant isoforms in these cells. In contrast to other neuronal cell lines, none of these isoforms is down-regulated during differentiation. Confocal immunofluorescence microscopy revealed that in undifferentiated cells PKC alpha is located in the cytoplasm and in the nucleus excluding nucleoli. In differentiated cells PKC alpha was almost exclusively located in the cytoplasm. Stimulation of the cells with phorbol ester resulted in translocation to the plasma membrane. PKC beta II was not detectable in the nuclei. PKC delta was found in the nucleoli and in the cytoplasm, in differentiated cells particularly in the neurites. Phorbol ester failed to induce a translocation to other compartments. PKC epsilon was localized with the nuclear-pore complexes at the nuclear envelope. In differentiated cells after stimulation with phorbol ester, partial translocation to the plasma membrane was observed.

Using isoenzyme-specific antisera, protein kinase C (PKC) alpha and PKC delta were detected in total liver homogenate and in isolated nuclei. PKC beta I, beta II, epsilon, epsilon', and zeta were not detected. During liver regeneration, nuclear PKC alpha levels decreased while PKC delta levels increased. These studies demonstrate, for the first time, the presence of a calcium-independent PKC isoenzyme in liver nuclei and suggest that PKC alpha and PKC delta may have different roles in liver regeneration and cell proliferation.

The promyelocytic leukemia (HL60) cell line differentiates into monocyte-like cells after treatment with phorbol dibutyrate (PBt2). In contrast, bryostatin 1 (bryo), a structurally distinct protein kinase C (PKC) activator, does not induce differentiation and blocks the cytostatic effect of PBt2. The divergent responses to these agents correlate with activation of a PKC-like activity at the nucleus in response to bryo but not PBt2 (Fields, A. P., Pettit, G. R., and May, W.S. (1988) J. Biol. Chem. 263, 8253-8260). In the present study, this nuclear PKC-like activity (termed PKCn) was isolated from HL60 cells and shown to phosphorylate its known nuclear substrate, lamin B. PKCn-mediated phosphorylation of nuclear envelope-associated lamin B in vitro is calcium-dependent and is stimulated by bryo and 1,2-dioctanoylglycerol (DiC8), but not PBt2. In contrast, PKCn-mediated phosphorylation of histone IIIS is stimulated equally by all three activators. PKCn mediates calcium- and phosphatidylserine-dependent phosphorylation of both histone IIIS and partially purified lamin B. PKCn activity can be inhibited by an anti-PKC monoclonal antibody which specifically inhibits PKC. Isotype-specific PKC antibodies identify PKCn as beta II-PKC. Immunoblot analysis indicates that HL60 cells express both alpha- and beta II-PKC but no beta I- or gamma-PKC. Treatment of intact cells with bryo for 30 min leads to complete translocation of both alpha- and beta II-PKC from the cytosol to the membrane fractions. Approximately 8-10% of the total beta II-PKC (and less than 0.3% of the alpha-PKC) is found associated with the nuclear membrane of bryo-treated cells. In contrast, PBt2 treatment leads to complete translocation of alpha-PKC, but only partial translocation of beta II-PKC to the plasma membrane fraction. Neither PKC isotype is found associated with the nuclear membrane of PBt2-treated cells. These data demonstrate that alpha- and beta II-PKC differ with respect to activator responsiveness, intracellular distribution, and substrate specificity and indicate that their selective activation at distinct intracellular sites, including the nucleus, can have a dramatic effect on resulting cellular responses.

Procedures for isolation, from human term placenta, of highly purified nuclei and nuclear envelopes with a low content of DNA are described. Both fractions contain oestrone sulphate sulphohydrolase activity. The enzyme from nuclear envelopes can be solubilized with Triton X-100 and, partially, with proteolytic enzymes. It does not require Ca2+ and is insensitive to Ag+ and agents reacting with SH groups. It is strongly inhibited by millimolar concentrations of sulphites and to a much smaller extent by phosphates. Oxidized forms of ascorbic acid, glutathione and NAD+ revealed a pronounced inhibitory effect, whereas reduced forms of these compounds produced a slight activation. It is proposed that oestrone sulphate sulphohydrolase activity in nuclear envelopes from human placenta is not exerted by arylsulphatase but represents a specific enzyme.

It seems well established that translocation of at least some mRNAs through the nuclear pore is (1) an energy-dependent process, and (2) dependent on the presence of the poly(A) segment attached to most mRNA species. We describe that RNA helicase (RNA duplex unwindase) activity is present in a nuclear envelope (NE) preparation, which also appears to be involved in nucleocytoplasmic RNA transport. This activity unwinds RNA: RNA hybrids. The helicase has a pH optimum of 7.5 and a temperature optimum of 30 degrees C. Applying the sealed NE vesicle system, it was shown that duplex RNA species are readily released from the vesicles in an unidirectional manner, in contrast to single-stranded RNA, which is much slower transported into the extravesicular space. Attachment of a poly(A) segment to the RNA duplex additionally increases the efflux rate of this RNA. Efflux of duplex RNA but not efflux of single-stranded RNA was strongly inhibited by formycin B 5'-triphosphate. Our results suggest that, besides poly(A), duplex structures, if present in a given RNA, modulate and control the export of RNA.

The efficiency of efflux of rapidly labeled poly(A)-containing mRNA from isolated rat liver nuclei was found to be modulated by insulin and epidermal growth factor (EGF) in a biphasic but opposite way. At physiological concentrations (10 pM insulin and 1 pM EGF), maximal stimulation of the transport rate by insulin (to 137%) and maximal inhibition by EGF (to 69%) were obtained; at higher concentrations (greater than 100 pM and greater than 10 pM, respectively), the amount of poly(A)-containing mRNA released into the postnuclear supernatant was nearly identical with the level found in untreated nuclei (= 100%). Using mRNA entrapped into closed nuclear envelope (NE) vesicles as a model system, it was found that the modulation of nuclear efflux of mRNA by the two growth factors occurs at the level of translocation through the nuclear pore. The NE nucleoside-triphosphatase (NTPase) activity, which is thought to mediate nucleocytoplasmic transport of at least some mRNAs, responded to insulin and EGF in the same manner as the mRNA transport rate. The increase in NTPase activity caused by insulin and the decrease in NTPase activity caused by EGF were found to be due to changes of the maximal catalytic rate; the Michaelis constant of the enzyme remained almost constant. Investigating the effect of the two growth factors on transport of specific mRNAs, poly(A)-containing actin mRNA was found to display the same alteration in efflux rate as rapidly labeled, total poly(A)-containing mRNA. In contrast, efflux of histone H4 mRNA, which lacks a 3'-poly(A) sequence, decreased in response to insulin and reached minimum levels at the same concentration at which maximum levels of actin mRNA transport rate were obtained. Studying the mechanism of action of insulin and EGF on NE mRNA translocation system, insulin was found to cause an enhancement of NE-associated phosphoprotein phosphatase activity, resulting in a dephosphorylation of the NE poly(A) binding site (= mRNA carrier) and, hence, in a decrease in its affinity to poly(A) [the poly(A) binding affinity of the poly(A)-recognizing mRNA carrier within the envelope is increased after phosphorylation]. EGF, on the other hand, stimulated the protein kinase, which phosphorylates the carrier, and, hence increased the NE poly(A) binding affinity. Because the stage of phosphorylation of the mRNA carrier (which is coupled with the NTPase within the intact NE structure) is inversely correlated with the activity of the NTPase, an enhancement of poly(A)-containing mRNA transport rate by insulin and an inhibition by EGF are observed.

A comparative study of glucose-6-phosphatase, alcaline RNase, ATPase, inosine diphosphatase and 5'-nucleotidase activities in isolated rat liver and hepatoma-27 nuclei and nuclear envelopes was performed. The tumor nuclear membranes were shown to be free from G-6-Pase activity in contrast to the liver nuclear membranes. The nuclear RNase activity was strongly inhibited in the hepatoma and could be unmasked in the presence of 3-10(-4) M pCMB. Hepatoma nuclear and nuclear envelopes ATP-ase activity was found to be moderately decreased as compared to those of the normal tissue. The values of inosine diphosphatase activity in hepatoma were similar to those in liver. The role of the nuclear envelope in nuclear-cytoplasmic interactions as well as nuclear location of G-6-Pase are discussed.

The intracellular location of the enzymes that excise tRNA intervening sequences and subsequently religate the molecular was investigated in Xenopus oocytes. All the splicing activity detected in a whole oocyte was present in the nucleus. By manual separation of the nuclear envelope and the nuclear contents (which form a gel-like structure in the presence of Mg++) it was found that all the activity of both splicing enzymes was located in the nuclear contents and was not associated with the nuclear membrane.

Yeast tRNA ligase is an enzyme required for tRNA splicing. A study by indirect immune fluorescence shows that this enzyme is localized in the cell nucleus. At higher resolution, studies using indirect immune electron microscopy show this nuclear location to be primarily at the inner membrane of the nuclear envelope, most likely at the nuclear pore. There is a more diffuse, secondary location of ligase in a region of the nucleoplasm within 300 nm of the nuclear envelope. When the amount of ligase in the cell is increased, nuclear staining increases but staining of the nuclear envelope remains constant. This experiment indicates that there are a limited number of ligase sites at the nuclear envelope. Since the other tRNA splicing component, the endonuclease, has the characteristics of an integral membrane protein, we hypothesize that it constitutes the site for the interaction of ligase with the nuclear envelope.

After infection of the respective target cells with the human immunodeficiency virus (HIV-1) viral progeny is produced only after a short temporary delay of some days, depending on cell type. After this period of time a sudden onset of HIV-1 protein synthesis with a dramatic increase in virus release occurs. (2'-5')Oligoriboadenylates [(2'-5')A], capable to activate a latent ribonuclease (RNase L) degrading both mRNA and rRNA, are known mediators involved in the early response of cells to virus infection. Here we show that the (2'-5')A-synthesizing (2'-5')A synthetase, which is inducible by interferon and activated by double-stranded RNA, as well as a (2'-5')A nuclease (2',3'-exoribonuclease) are associated with the nuclear matrix of uninfected and infected H9 cells, also in the absence of interferon. Infection of H9 cells with HIV-1 was found to cause a strong (7.7-fold) enhancement of (2'-5')A synthetase activity and a smaller (2-fold) increase of 2',3'-exoribonuclease activity. Simultaneously the concentration of synthesized (2'-5')A increased 5 to 10 times in isolated nuclei. After incubation for 2 to 3 days both enzyme activities reached a maximum and then dropped below their initial values. Concomitantly a drastic increase in virus production occurred, as judged by reverse transcriptase activity in the culture fluid. These results suggest that the (nuclear matrix-associated) (2'-5')A system might be important during the initial stage of HIV infection, also by destructing matrix-bound viral messengers.