Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells.

BACKGROUND: It has been found that chronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC). CRS can be caused by gastro-oesophageal reflux (GOR) that may reach nasopharynx. The major component of refluxate, bile acid (BA) has been found to be carcinogenic and genotoxic. BA-induced apoptosis has been associated with various cancers. We have previously demonstrated that BA induced apoptosis and gene cleavages in nasopharyngeal epithelial cells. Chromosomal cleavage occurs at the early stage of both apoptosis and chromosome rearrangement. It was suggested that chromosome breaks tend to cluster in the region containing matrix association region/scaffold attachment region (MAR/SAR). This study hypothesised that BA may cause chromosome breaks at MAR/SAR leading to chromosome aberrations in NPC. This study targeted the AF9 gene located at 9p22 because 9p22 is a deletion hotspot in NPC. METHODS: Potential MAR/SAR sites were predicted in the AF9 gene by using MAR/SAR prediction tools. Normal nasopharyngeal epithelial cells (NP69) and NPC cells (TWO4) were treated with BA at neutral and acidic pH. Inverse-PCR (IPCR) was used to identify chromosome breaks in SAR region (contains MAR/SAR) and non-SAR region (does not contain MAR/SAR). To map the chromosomal breakpoints within the AF9 SAR and non-SAR regions, DNA sequencing was performed. RESULTS: In the AF9 SAR region, the gene cleavage frequencies of BA-treated NP69 and TWO4 cells were significantly higher than those of untreated control. As for the AF9 non-SAR region, no significant difference in cleavage frequency was detected between untreated and BA-treated cells. A few breakpoints detected in the SAR region were mapped within the AF9 region that was previously reported to translocate with the mixed lineage leukaemia (MLL) gene in an acute lymphoblastic leukaemia (ALL) patient. CONCLUSIONS: Our findings suggest that MAR/SAR may be involved in defining the positions of chromosomal breakages induced by BA. Our report here, for the first time, unravelled the relation of these BA-induced chromosomal breakages to the AF9 chromatin structure.

PDGFRß translocates to the nucleus and regulates chromatin remodeling via TATA element-modifying factor 1.

Translocation of full-length or fragments of receptors to the nucleus has been reported for several tyrosine kinase receptors. In this paper, we show that a fraction of full-length cell surface platelet-derived growth factor (PDGF) receptor ß (PDGFRß) accumulates in the nucleus at the chromatin and the nuclear matrix after ligand stimulation. Nuclear translocation of PDGFRß was dependent on PDGF-BB-induced receptor dimerization, clathrin-mediated endocytosis, ß-importin, and intact Golgi, occurring in both normal and cancer cells. In the nucleus, PDGFRß formed ligand-inducible complexes with the tyrosine kinase Fer and its substrate, TATA element-modifying factor 1 (TMF-1). PDGF-BB stimulation decreased TMF-1 binding to the transcriptional regulator Brahma-related gene 1 (Brg-1) and released Brg-1 from the SWI-SNF chromatin remodeling complex. Moreover, knockdown of TMF-1 by small interfering RNA decreased nuclear translocation of PDGFRß and caused significant up-regulation of the Brg-1/p53-regulated cell cycle inhibitor CDKN1A (encoding p21) without affecting PDGFRß-inducible immediate-early genes. In conclusion, nuclear interactions of PDGFRß control proliferation by chromatin remodeling and regulation of p21 levels.

The vitamin D receptor (VDR) binds to the nuclear matrix via its hinge domain: A potential mechanism for the reduction in VDR mediated transcription in mitotic cells.

Vitamin D is best known for its regulation of calcium homeostasis. Vitamin D exerts its genomic actions via the vitamin D receptor (VDR). As a member of the superfamily of nuclear receptors (NR), the VDR is primarily located within the nucleus of non-dividing cells. We show here that the VDR relocates from the nucleus into the cytoplasm across all stages of cell division in CHO cells. Furthermore, we show that the VDR is transcriptionally inert during cell division. In addition, 1α, 25 dihydroxyvitamin D (1,25(OH)2D3) promotes VDR binding to the nuclear matrix. Finally, we assessed the structural nature of VDR binding to the nuclear matrix. Mutation of the hinge domain reduced VDR's ability to bind to the nuclear matrix and to initiate transcription in response to 1,25(OH)2D3. Taken together, our data suggest that the association between the VDR and the nuclear matrix accounts for the apparent cytosolic distribution as the matrix disperses within the cytoplasm when cells divide. This may also explain the dramatic reduction in VDR mediated transcription during cell division. Our data also confirm that similar to other NRs, the hinge domain of the VDR is responsible for this association.

Analysis of quinolinequinone reactivity, cytotoxicity, and anti-HIV-1 properties.

We have analyzed a set of quinolinequinones with respect to their reactivities, cytotoxicities, and anti-HIV-1 properties. Most of the quinolinequinones were reactive with glutathione, and several acted as sulfhydryl crosslinking agents. Quinolinequinones inhibited binding of the HIV-1 matrix protein to RNA to varying degrees, and several quinolinequinones showed the capacity to crosslink HIV-1 matrix proteins in vitro, and HIV-1 structural proteins in virus particles. Cytotoxicity assays yielded quinolinequinone CC50 values in the low micromolar range, reducing the potential therapeutic value of these compounds. However, one compound, 6,7-dichloro-5,8-quinolinequinone potently inactivated HIV-1, suggesting that quinolinequinones may prove useful in the preparation of inactivated virus vaccines or for other virucidal purposes.

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication.

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.

Double-stranded DNA breaks hidden in the neutral Comet assay suggest a role of the sperm nuclear matrix in DNA integrity maintenance.

We used a mouse model in which sperm DNA damage was induced to understand the relationship of double-stranded DNA (dsDNA) breaks to sperm chromatin structure and to the Comet assay. Sperm chromatin fragmentation (SCF) produces dsDNA breaks located on the matrix attachment regions, between protamine toroids. In this model, epididymal sperm induced to undergo SCF can religate dsDNA breaks while vas deferens sperm cannot. Here, we demonstrated that the conventional neutral Comet assay underestimates the epididymal SCF breaks because the broken DNA ends remain attached to the nuclear matrix, causing the DNA to remain associated with the dispersion halo, and the Comet tails to be weak. Therefore, we term these hidden dsDNA breaks. When the Comet assay was modified to include an additional incubation with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) after the conventional lysis, thereby solubilizing the nuclear matrix, the broken DNA was released from the matrix, which resulted in a reduction of the sperm head halo and an increase in the Comet tail length, exposing the hidden dsDNA breaks. Conversely, SCF-induced vas deferens sperm had small halos and long tails with the conventional neutral Comet assay, suggesting that the broken DNA ends were not tethered to the nuclear matrix. These results suggest that the attachment to the nuclear matrix is crucial for the religation of SCF-induced DNA breaks in sperm. Our data suggest that the neutral Comet assay identifies only dsDNA breaks that are released from the nuclear matrix and that the addition of an SDS treatment can reveal these hidden dsDNA breaks.

Roles of the intramolecular regions of FE65 in its trans-accumulation and in p53 stabilization in the nuclear matrix of osmotically stressed cells.

The neural adaptor protein FE65 interacts with the amyloid beta-protein precursor (APP). In osmotically stressed cells, the membrane APP-tethered FE65 is released into the cytoplasm and translocates to the nuclear matrix, where it stabilizes p53 via a non-canonical pathway. In this study, we found that the second phosphotyrosine interaction domain (PI2) of FE65 mediated its trans-accumulation in the nuclear matrix of osmotically stressed cells. The carboxyl-terminal half of FE65, which contains the PI2 domain, failed to stabilize p53, suggesting that the amino-terminal half of the protein plays an important role in the stabilization of p53 in osmotically stressed cells.

Localization of X-ray cross complementing gene 1 protein in the nuclear matrix is controlled by casein kinase II-dependent phosphorylation in response to oxidative damage.

Base excision repair/single strand break repair (BER/SSBR) of damaged DNA is a highly efficient process. X-ray cross complementing protein 1 (XRCC1) functions as a key scaffold protein for BER/SSBR factors. Recent work has shown that XRCC1 forms dense foci at sites of DNA damage in a manner dependent on casein kinase II (CK2) phosphorylation. To investigate the mechanism underlying foci formation, we analyzed the subnuclear localization and phosphorylation status of XRCC1 during the repair process by biochemical fractionation of HeLa cellular proteins. The localization was also verified by in situ extraction of the fixed cells. In unchallenged cells, XRCC1 was primarily found in the chromatin fraction in a highly phosphorylated form; in addition, a minor population (10-15%) existed in the nuclear matrix (NM) with no or marginal phosphorylation. After hydrogen peroxide treatment, hyperphosphorylated XRCC1 appeared in the NM and accordingly, those in the chromatin fraction decreased. Foci formation and changes in XRCC1 distribution could be abolished by the knockdown of CK2, the expression of a non-phosphorylatable version of XRCC1, or the inhibition of poly-ADP ribosylation at the damage sites. Other BER factors, like DNA polymerase beta, were also found to accumulate in the NM after hydrogen peroxide-induced DNA damage, although its association with the NM seemed relatively weak. Our results suggest that the constitutive phosphorylation of XRCC1 in the chromatin and its DNA damage-induced recruitment to the NM are critical for foci formation, and that the core reactions of BER/SSBR may occur in the NM.

Epidermal growth factor stimulates translocation of the class II phosphoinositide 3-kinase PI3K-C2beta to the nucleus.

Although the class II phosphoinositide 3-kinase enzymes PI3K-C2alpha and PI3K-C2beta act acutely downstream of cell surface receptors they have also been localized to nuclei in mammalian cells. As with the class I PI3K enzymes, the relationship between the pools of enzyme present in cytoplasm and nuclei remains poorly understood. In this study we test the hypothesis that PI3K-C2beta translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2beta resides in nuclei. Stimulation with epidermal growth factor sequentially increased levels of this enzyme, firstly in the cytosol and secondly in the nuclei. Using detergent-treated nuclei, we showed that PI3K-C2beta co-localized with lamin A/C in the nuclear matrix. This was confirmed biochemically, and a phosphoinositide kinase assay showed a statistically significant increase in nuclear PI3K-C2beta levels and lipid kinase activity following epidermal growth factor stimulation. C-terminal deletion and point mutations of PI3K-C2beta demonstrated that epidermal growth factor-driven translocation to the nucleus is dependent on a sequence of basic amino acid residues (KxKxK) that form a nuclear localization motif within the C-terminal C2 domain. Furthermore, when this sequence was expressed as an EGFP (enhanced green fluorescent protein) fusion protein, it translocated fluorescence into nuclei with an efficiency dependent upon copy number. These data demonstrate that epidermal growth factor stimulates the appearance of PI3K-C2beta in nuclei. Further, this effect is dependent on a nuclear localization signal present within the C-terminal C2 domain, indicating its bimodal function regulating phospholipid binding and shuttling PI3K-C2beta into the nucleus.

[Localization of prohibitin in nuclear matrix and its expressive alteration during the differentiation of human osteosarcoma MG-63 cells induced by HMBA].

To explore the existence,distribution of prohibitin in nuclear matrix and the co-localization relationship between prohibitin and the products of some interrelated genes in the human osteosarcoma MG-63 cells before and after HMBA treatment, the nuclear matrix of MG-63 cells and the cells induced by HMBA were selectively extracted. It was confirmed by Western blot that prohibitin existed in the component of nuclear matrix protein of MG-63 cells and its expression was decreased by HMBA treatment. The immunofluorescence observation revealed that prohibitin located in the nuclear matrix, and its distribution regions and expression level had altered after HMBA treatment. The co-localization and its alternation of distributive area in the cells treated by HMBA were observed between prohibitin and the products of oncogenes or tumor repression genes including c-fos, c-myc, p53 and Rb by using laser scanning confocal microscopy. The results of this study demonstrated that prohibitin was a kind of nuclear matrix protein, and locacted in the nuclear matrix,and the distribution and expression of prohibitin and its relationship with associated genes play an important role during the differentiation of MG-63 cell.

Alteration of nuclear matrix-intermediate filament system and differential expression of nuclear matrix proteins during human hepatocarcinoma cell differentiation.

AIM: To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells. METHODS: Cells cultured with or without 5 x 10(-3) mmol/L of hexamethylene bisacetamide (HMBA) on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin. The peptides were analyzed by matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Data were submitted for database searching using Mascot tool (www.matrixscience.com). RESULTS: The nuclear matrix (NM) and intermediate filament (IF) in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly. The nuclear lamina was non-uniform, and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA, the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments, including highly branched ultrathin filaments could also be seen in the regular meshwork. The connection between the two kinds of filaments and the relatively thin, condensed and sharply demarcated lamina composed of intermediate-sized filaments was relatively fastened. Meanwhile, 21 NM proteins changed remarkably during SMMC-7721 cell differentiation. Four proteins, i.e. mutant Pyst1, hypothetical protein, nucleophosmin 1, and LBP were downregulated, whereas four other proteins, eIF6, p44 subunit, beta-tubulin, and SIN3B were upregulated with the last one, SR2/ASF found only in the differentiated SMMC-7721 cells. CONCLUSION: The induced differentiation of SMMC-7721 cells by HMBA is accompanied by the configurational changes of nuclear matrix-intermediate filament (NM-IF) system and the compositional changes of nuclear matrix protein expression. These changes may be important morphological or functional indications of the cancer cell reversion.

Chronic cyclophosphamide exposure alters the profile of rat sperm nuclear matrix proteins.

Chronic exposure of male rats to the alkylating agent cyclophosphamide, a well-known male-mediated developmental toxicant, alters gene expression in male germ cells as well as in early preimplantation embryos sired by cyclophosphamide-exposed males. Sperm DNA is organized by the nuclear matrix into loop-domains in a sequence-specific manner. In somatic cells, loop-domain organization is involved in gene regulation. Various structural and functional components of the nuclear matrix are targets for chemotherapeutic agents. Consequently, we hypothesized that cyclophosphamide treatment would alter the expression of sperm nuclear matrix proteins. Adult male rats were treated for 4 wk with saline or cyclophosphamide (6.0 mg kg(-1) day(-1)), and the nuclear matrix was extracted from cauda epididymal sperm. Proteins were analyzed by two-dimensional gel electrophoresis. Identified proteins within the nuclear matrix proteome were mainly involved in cell structure, transcription, translation, DNA binding, protein processing, signal transduction, metabolism, cell defense, or detoxification. Interestingly, cyclophosphamide selectively induced numerous changes in cell defense and detoxification proteins, most notably, in all known forms of the antioxidant enzyme glutathione peroxidase 4, in addition to an uncharacterized 54-kDa form; an overall increase in glutathione peroxidase 4 immunoreactivity was observed in the nuclear matrix extracts from cyclophosphamide-exposed spermatozoa. An increase in glutathione peroxidase 4 expression suggests a role for this enzyme in maintaining nuclear matrix stability and function. These results led us to propose that a change in composition of the nuclear matrix in response to drug exposure was a factor in altered sperm function and embryo development.

Nur77 forms novel nuclear structures upon DNA damage that cause transcriptional arrest.

The orphan nuclear receptor Nur77 has been implicated in both growth and apoptosis, and its function and activity can be modulated by cellular redistribution. Green fluorescent protein-tagged Nur77 was used to evaluate the role of Nur77 intracellular redistribution in response to genotoxic stress. Selected DNA damaging agents and transcription inhibition lead to rapid redistribution of Nur77 into nuclear structures distinct from conventional nuclear bodies. These nuclear bodies formed transiently were tightly bound to the nuclear matrix and conditions that lead to their appearance were associated with Nur77 transcriptional inhibition. The formation of Nur77 nuclear bodies might be involved in programmed cell death modulation upon exposure to DNA damaging agents that inhibit transcription by sequestrating this proapoptotic factor in dense nuclear structures.

Assembly of nuclear matrix-bound protein complexes involved in non-homologous end joining is induced by inhibition of DNA topoisomerase II.

Topoisomerases maintain the DNA structure by relieving the torsional stress and alleviating other topological problems occurring in DNA during transcription and replication. Topoisomerase II appears to have a close association with the family of proteins involved in the organization of chromatin in a series of loops on the proteinaceous chromosomal matrix. Beyond its physiological functions, topoisomerase II is the target for some of the most active anticancer drugs. Inhibition of the topoisomerase II function can result in DNA double-strand breaks (DSBs) and, thus, lead to chromosomal translocations. The earliest event during DSB repair is phosphorylation of histone H2AX at S139 (so-called gammaH2AX) which is believed to serve as a focal point for the assembly of repair proteins at the DSB. In this work, we have demonstrated the formation of gammaH2AX foci in two human cell lines--K562 and HeLa--after suppression of topoisomerase II activity with etoposide. Furthermore, these foci remained visible at nuclear matrices and colocalized with the major components of non-homologous end joining (NHEJ) system of DSBs repair. Thus, inhibition of topoisomerase II activity triggers assembly of NHEJ complexes at the nuclear matrix.

Merbarone induces activation of caspase-activated DNase and excision of chromosomal DNA loops from the nuclear matrix.

Studies were carried out to address possible cellular mechanisms by which merbarone, a catalytic inhibitor of DNA topoisomerase II, can block tumor cell growth without inducing extensive DNA cleavage. Merbarone induced the release of high molecular weight DNA fragments from the nuclear matrix of HL-60 leukemia cells, which preceded the internucleosomalsize DNA fragmentation characteristic of late-stage apoptosis. The chromatin fragments were enriched in a matrix attachment region (MAR) sequence compared with a non-MAR sequence and were similar in size to DNA loops extracted from nuclear matrices. However, merbarone did not directly induce the excision of high molecular weight DNA fragments from the nuclear matrix by promoting topoisomerase II-catalyzed DNA cleavage, because the drug inhibited topoisomerase II-mediated cleavage in isolated nuclear matrix preparations. Instead, merbarone induced rapid activation of the mitochondrial apoptosis pathway, which included the following temporal sequence of events: dissipation of the mitochondrial transmembrane potential within 30 min, release of mitochondrial cytochrome c, and activation of caspase-activated DNase (CAD) by its inhibitor ICAD. The excision of high molecular weight DNA was inhibited at least 80% in merbarone-treated cells preincubated with the pan-caspase inhibitor z-VAD-fmk [Z-Val-Ala-Asp(OMe)-fluoromethyl ketone] and in caspase-resistant Jurkat cells (ICAD/double-mutated) that express a mutant form of ICAD. These results provide evidence that merbarone can induce rapid disorganization of DNA in tumor cells that have a functional mitochondrial apoptosis pathway without inducing extensive DNA cleavage.

Ca2+-dependent nuclear contraction in the heliozoon Actinophrys sol.

Ca2+-dependent contractility was found to exist in the nucleus of the heliozoon protozoan Actinophrys sol. Upon addition of Ca2+ ([Ca2+]free = 2.0 x 10(-3) M), diameters of isolated and detergent-extracted nuclei became reduced from 16.5+/-1.7 microm to 11.0+/-1.3 microm. The threshold level of [Ca2+]free for the nuclear contraction was 2.9 x 10(-7) M. The nuclear contraction was not induced by Mg2+, and was not inhibited by colchicine or cytochalasin B. Contracted nuclei became expanded when Ca2+ was removed by EGTA; thus cycles of contraction and expansion could be repeated many times by alternating addition of Ca2+ and EGTA. The Ca2+-dependent nuclear contractility remained even after high salt treatment, suggesting a possible involvement of nucleoskeletal components in the nuclear contraction. Electron microscopy showed that, in the relaxed state, filamentous structures were observed to spread in the nucleus to form a network. After addition of Ca2+, they became aggregated and constructed a mass of thicker filaments, followed by re-distribution of the filaments spread around inside of the nucleus when Ca2+ was removed. These results suggest that the nuclear contraction is induced by Ca2+-dependent transformation of the filamentous structures in the nucleus.

Diazinon alters sperm chromatin structure in mice by phosphorylating nuclear protamines.

Organophosphorus (OP) pesticides, widely used in agriculture and pest control, are associated with male reproductive effects, including sperm chromatin alterations, but the mechanisms underlying these effects are unknown. The main toxic action of OP is related to phosphorylation of proteins. Chemical alterations in sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis, contribute to male reproductive toxicity. Therefore, in the present study, we tested the ability of diazinon (DZN), an OP compound, to alter sperm chromatin by phosphorylating nuclear protamines. Mice were injected with a single dose of DZN (8.12 mg/kg, i.p.), and killed 8 and 15 days after treatment. Quality of sperm from epididymis and vas deferens was evaluated through standard methods and chromatin condensation by flow cytometry (DNA Fragmented Index parameters: DFI and DFI%) and fluorescence microscopy using chromomycin-A(3) (CMA(3)). Increases in DFI (15%), DFI% (4.5-fold), and CMA(3) (2-fold) were observed only at 8 days post-treatment, indicating an alteration in sperm chromatin condensation and DNA damage during late spermatid differentiation. In addition, an increase of phosphorous content (approximately 50%) in protamines, especially in the phosphoserine content (approximately 73%), was found at 8 days post-treatment. Sperm viability, motility, and morphology showed significant alterations at this time. These data strongly suggest that spermatozoa exposed during the late steps of maturation were the targets of DZN exposure. The correlation observed between the phosphorous content in nuclear protamines with DFI%, DFI, and CMA(3) provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin.

Gene positional changes relative to the nuclear substructure during carbon tetrachloride-induced hepatic fibrosis in rats.

In the interphase nucleus the DNA of higher eukaryotes is organized in loops anchored to a substructure known as the nuclear matrix (NM). The topological relationship between gene sequences located in the DNA loops and the NM appears to be very important for nuclear physiology because processes such as replication, transcription, and processing of primary transcripts occur at macromolecular complexes located at discrete sites upon the NM. Mammalian hepatocytes rarely divide but preserve a proliferating capacity that is displayed in vivo after specific stimulus. We have previously shown that transient changes in the relative position of specific genes to the NM occur during the process of liver regeneration after partial ablation of the liver, but also that such changes correlate with the replicating status of the cells. Moreover, since chronic exposure to carbon tetrachloride (CCl4) leads to bouts of hepatocyte damage and regeneration, and eventually to non-reversible liver fibrosis in the rat, we used this animal model in order to explore if genes that show differential activity in the liver change or modify their relative position to the NM during the process of liver fibrosis induction. We found that changes in the relative position of specific genes to the NM occur during the chronic administration of CCl4, but also that such changes correlate with the proliferating status of the hepatocytes that goes from quiescence to regeneration to replicative senescence along the course of CCl4-induced liver fibrosis, indicating that specific configurations in the higher-order DNA structure underlie the stages of progression towards liver fibrosis.

Nuclear diacylglycerol kinase-theta is activated in response to nerve growth factor stimulation of PC12 cells.

Previous evidence from independent laboratories has shown that the nucleus contains diacylglycerol kinase (DGK) isoforms, i.e., the enzymes, which yield phosphatidic acid from diacylglycerol, thus terminating protein kinase C-mediated signaling events. A DGK isoform, which resides in the nucleus of PC12 cells, is DGK-theta. Here, we show that nerve growth factor (NGF) treatment of serum-starved PC12 cells results in the stimulation of both a cytoplasmic and a nuclear DGK activity. However, time course analysis shows that cytoplasmic DGK activity peaked earlier than its nuclear counterpart. While nuclear DGK activity was dramatically down-regulated by a monoclonal antibody known for selectively inhibiting DGK-theta, cytoplasmic DGK activity was not. Moreover, nuclear DGK activity was stimulated by phosphatidylserine, an anionic phospholipid that had no effect on cytoplasmic DGK activity. Upon NGF stimulation, the amount and the activity of DGK-theta, which was bound to the insoluble nuclear matrix fraction, substantially increased. Epidermal growth factor up-regulated a nuclear DGK activity insensitive to anti-DGK-theta monoclonal antibody. Overall, our findings identify nuclear DGK-theta as a down-stream target of NGF signaling in PC12 cells.