Nuclear relocation of DGKζ in cardiomyocytes under conditions of ischemia/reperfusion

Diacylglycerol (DG) and phosphatidic acid (PA) are generated under various conditions, such as ligand stimulation and several stresses. They serve as second messengers to respond to pathophysiological conditions. DG kinase (DGK) catalyzes DG to produce PA. It is regarded as a regulator of these lipid messengers. Previous studies show that DGKζ, a nuclear isozyme, translocates from the nucleus to the cytoplasm in hippocampal neurons under transient ischemia and never relocates to the nucleus after reperfusion. This study examined whether a similar phenomenon is observed in cardiomyocytes, which represent another type of postmitotic, terminally differentiated cell. We performed immunostaining on ischemic hearts induced by occlusion of the left anterior descending coronary artery and on primary cultured cardiomyocytes under oxygen-glucose deprivation (OGD). In the animal model, 10 min ischemia is sufficient to cause DGKζ to disappear from the nucleus in cardiomyocytes. However, DGKζ is observed again in the nucleus at 10 min following reperfusion after 10 min ischemia, which contrasts sharply with ischemic hippocampal neurons. Similar results were obtained from experiments using primary cultured cardiomyocytes under OGD conditions, except that DGKζ relocates autonomously, if at all, to the nucleus, even under continuous OGD conditions. Results suggest that DGKζ is involved in the acute phase of cellular response to ischemic stress in cardiomyocytes in a similar, but not identical, manner to that of neurons.     

Diacylglycerol Kinase Inhibition and Vascular Function

Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP(3)), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction.     

Regulation of Chk1 includes chromatin association and 14-3-3 binding following phosphorylation on Ser-345

Checkpoints are biochemical pathways that provide the cell with mechanisms to detect DNA damage and respond by arresting the cell cycle to allow DNA repair. The conserved checkpoint kinase Chk1 regulates mitotic progression in response to DNA damage and replication interference by blocking the activation of Cdk1/cyclin B. Chk1 is phosphorylated on Ser-317 and Ser-345 following a checkpoint signal, a process that is regulated by Atr, and by the sensor complexes containing Rad17 and Hus1. We show that Chk1 is associated with chromatin in cycling cells and that the chromatin-associated Chk1 is phosphorylated in the absence of exogenous DNA damage. The UV-induced Ser-345-phosphorylated forms of Chk1 that appear minutes after treatment are predominantly associated with chromatin. The Ser-345 site is in a 14-3-3 consensus binding motif and is required for nuclear retention of Chk1 following an hydroxyurea-induced checkpoint signal; nonetheless, Ser-345 or Ser-317 are not required for the chromatin association of Chk1. Hus1, a member of the proliferating cell nuclear antigen-like damage recognition complex plays a role in the phosphorylation of Chk1 on Ser-345, however, Hus1 is not required for phosphorylation on Ser-317 or for Chk1 localization to chromatin. These results indicate that there is more than one step in Chk1 activation and that the regulation of this checkpoint signaling is achieved at least in part through phosphorylation of Ser-345, which serves to localize Chk1 in the nucleus presumably by blocking Crm1-dependent nuclear export.     

Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance

The initiation of DNA replication in S phase requires the prior assembly of an origin recognition complex (ORC)-dependent pre-replicative complex on chromatin during G1 phase of the cell division cycle. In human cells, the Orc2 subunit localized to the nucleus as expected, but it also localized to centrosomes throughout the entire cell cycle. Furthermore, Orc2 was tightly bound to heterochromatin and heterochromatin protein 1alpha (HP1alpha) and HP1beta in G1 and early S phase, but during late S, G2 and M phases tight chromatin association was restricted to centromeres. Depletion of Orc2 by siRNA caused multiple phenotypes. A population of cells showed an S-phase defect with little proliferating cell nuclear antigen (PCNA) on chromatin, although MCM proteins remained. Orc2 depletion also disrupted HP1 localization, but not histone-H3-lysine-9 methylation at prominent heterochromatic foci. Another subset of Orc2-depleted cells containing replicated DNA arrested with abnormally condensed chromosomes, failed chromosome congression and multiple centrosomes. These results implicate Orc2 protein in chromosome duplication, chromosome structure and centrosome copy number control, suggesting that it coordinates all stages of the chromosome inheritance cycle.     

Differential subcellular targeting and activity-dependent subcellular localization of diacylglycerol kinase isozymes in transfected cells

Diacylglycerol kinase (DGK) plays a pivotal role in cellular signal transduction through regulating levels of the second messenger diacylglycerol (DG). Previous studies have revealed that DGK is composed of a family of isozymes that show remarkable heterogeneity in terms of molecular structure, functional domains, tissue and cellular gene expression. Recently, it has been shown that DG is produced in various subcellular compartments including the plasma membrane, internal membranes, cytoskeleton, and nucleus. However, it remains unclear how DG is regulated at distinct subcellular sites. To address this point, we have used an epitope-tag expression system in cultured cells and investigated the subcellular localization of DGK isozymes under the same experimental conditions. We show here that DGK isozymes are targeted differentially to unique subcellular sites in transfected COS7 cells, including the cytoplasm, actin stress fibers, Golgi complex, endoplasmic reticulum, and nucleus. It is also shown that among the isozymes overexpression of DGKbeta causes fragmentation of actin stress fibers while a kinase-dead mutant of DGKbeta abolishes its colocalization with actin stress fibers. These data strongly suggest that each isozyme may be responsible for the metabolism of DG that is produced upon stimulation at a different and specific subcellular site and that DGKbeta activity might have effects on the reorganization of actin stress fibers in transfected COS7 cells.     

An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth

Changes in nuclear size and shape during the cell cycle or during development require coordinated nuclear membrane remodeling, but the underlying molecular events are largely unknown. We have shown previously that the activity of the conserved phosphatidate phosphatase Pah1p/Smp2p regulates nuclear structure in yeast by controlling phospholipid synthesis and membrane biogenesis at the nuclear envelope. Two screens for novel regulators of phosphatidate led to the identification of DGK1. We show that Dgk1p is a unique diacylglycerol kinase that uses CTP, instead of ATP, to generate phosphatidate. DGK1 counteracts the activity of PAH1 at the nuclear envelope by controlling phosphatidate levels. Overexpression of DGK1 causes the appearance of phosphatidate-enriched membranes around the nucleus and leads to its expansion, without proliferating the cortical endoplasmic reticulum membrane. Mutations that decrease phosphatidate levels decrease nuclear membrane growth in pah1Delta cells. We propose that phosphatidate metabolism is a critical factor determining nuclear structure by regulating nuclear membrane biogenesis.     

T-antigen interactions with chromatin and p53 during the cell cycle in extracts from xenopus eggs.

The role of SV40 large tumor T-antigen in replication of viral DNA is well established, but it is still unclear how T-antigen triggers cellular replication and cell transformation in non-permissive cells. Here, we used Xenopus egg extracts which reproduce most nuclear events linked to the cell cycle in vitro to analyze its interaction with genomic chromatin during the cell cycle. We show that T-antigen associates with chromatin before the nuclear membrane formation, and further demonstrate that the nuclear membrane is not necessary for its import into the nucleus. We show that the interaction of T-antigen with the endogenous chromatin does not occur at replication foci nor at RPA pre-replication centers. Immunoprecipitations as well as sucrose gradient experiments, indicate that the endogenous pool of p53 interacts with T-antigen. In addition, a transient association of both proteins with the nuclear matrix is observed during the ongoing DNA synthesis. These data are discussed in view of the T-antigen and p53 activity during the cell cycle.     

Nuclear transportation of diacylglycerol kinase gamma and its possible function in the nucleus

Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to phosphatidic acid, and both lipids are known to play important roles in lipid signal transduction. Thereby, DGKs are considered to be a one of the key players in lipid signaling, but its physiological function remains to be solved. In an effort to investigate one of nine subtypes, we found that DGKgamma came to be localized in the nucleus with time in all cell lines tested while seen only in the cytoplasm at the early stage of culture, indicating that DGKgamma is transported from the cytoplasm to the nucleus. The nuclear transportation of DGKgamma didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGKgamma acts as a nuclear transport signal. Furthermore, to address the function of DGKgamma in the nucleus, we produced stable cell lines of wild-type DGKgamma and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGKgamma were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGKgamma regulates cell cycle.     

The radial positioning of chromatin is not inherited through mitosis but is established de novo in early G1

The organization of chromatin in the nucleus is nonrandom. Different genomic regions tend to reside in preferred nuclear locations, relative to radial position and nuclear compartments. Several lines of evidence support a role for chromatin localization in the regulation of gene expression. Therefore, a key problem is how the organization of chromatin is established and maintained in dividing cell populations. There is controversy about the extent to which chromatin organization is inherited from mother to daughter nucleus. We have used time-lapse microscopy to track specific human loci after exit from mitosis. In comparison to later stages of interphase, we detect increased chromatin mobility during the first 2 hr of G1, and during this period association of loci with nuclear compartments is both gained and lost. Although chromatin in daughter nuclei has a rough symmetry in its spatial distribution, we show, for the first time, that the association of loci with nuclear compartments displays significant asymmetry between daughter nuclei and therefore cannot be inherited from the mother nucleus. We conclude that the organization of chromatin in the nucleus is not passed down precisely from one cell to its descendents but is more plastic and becomes refined during early G1.     

An extracellular signal-regulated kinase 1- and 2-dependent program of chromatin trafficking of c-Fos and Fra-1 is required for cyclin D1 expression during cell cycle reentry

Mitogens activate cell signaling and gene expression cascades that culminate in expression of cyclin D1 during the G(0)-to-G(1) transition of the cell cycle. Using cell cycle arrest in response to oxidative stress, we have delineated a dynamic program of chromatin trafficking of c-Fos and Fra-1 required for cyclin D1 expression during cell cycle reentry. In serum-stimulated lung epithelial cells, c-Fos was expressed, recruited to chromatin, phosphorylated at extracellular signal-regulated kinase 1- and 2 (ERK1,2)-dependent sites, and degraded prior to prolonged recruitment of Fra-1 to chromatin. Immunostaining showed that expression of nuclear c-Fos and that of cyclin D1 are mutually exclusive, whereas nuclear Fra-1 and cyclin D1 are coexpressed as cells traverse G(1). Oxidative stress prolonged the accumulation of phospho-ERK1,2 and phospho-c-Fos on chromatin, inhibited entry of Fra-1 into the nucleus, and blocked cyclin D1 expression. After induction of the immediate-early gene response in the presence of oxidative stress, inhibition of ERK1,2 signaling promoted degradation of c-Fos, recruitment of Fra-1 to chromatin, and expression of cyclin D1. Our data indicate that termination of nuclear ERK1,2 signaling is required for an exchange of Fra-1 for c-Fos on chromatin and initiation of cyclin D1 expression at the G(0)-to-G(1) transition of the cell cycle.     

Cell cycle-dependent and DNA damage-inducible nuclear localization of FEN-1 nuclease is consistent with its dual functions in DNA replication and repair

Flap endonuclease-1 (FEN-1), a 43-kDa protein, is a structure-specific and multifunctional nuclease. It plays important roles in RNA primer removal of Okazaki fragments during DNA replication, DNA base excision repair, and maintenance of genome stability. Three functional motifs of the enzyme were proposed to be responsible for its nuclease activities, interaction with proliferating cell nuclear antigen, and nuclear localization. In this study, we demonstrate in HeLa cells that a signal located at the C terminus (the nuclear localization signal (NLS) motif) facilitates nuclear localization of the enzyme during S phase of the cell cycle and in response to DNA damage. Truncation of the NLS motif prevents migration of the protein from the cytoplasm to the nucleus, while having no effect on the nuclease activities and its proliferating cell nuclear antigen interaction capability. Site-directed mutagenesis further revealed that a mutation of the KRK cluster to three alanine residues completely blocked the localization of FEN-1 into the nucleus, whereas mutagenesis of the KKK cluster led to a partial defect of nuclear localization in HeLa cells without observable phenotype in yeast. Therefore, the KRKXXXXXXXXKKK motif may be a bipartite NLS driving the protein into nuclei. Yeast RAD27Delta cells transformed with human mutant M(krk) survived poorly upon methyl methanesulfonate treatment or when they were incubated at an elevated temperature.     

Nuclear Translocation of hARD1 Contributes to Proper Cell Cycle Progression

Arrest defective 1 (ARD1) is an acetyltransferase that is highly conserved across organisms, from yeasts to humans. The high homology and widespread expression of ARD1 across multiple species and tissues signify that it serves a fundamental role in cells. Human ARD1 (hARD1) has been suggested to be involved in diverse biological processes, and its role in cell proliferation and cancer development has been recently drawing attention. However, the subcellular localization of ARD1 and its relevance to cellular function remain largely unknown. Here, we have demonstrated that hARD1 is imported to the nuclei of proliferating cells, especially during S phase. Nuclear localization signal (NLS)-deleted hARD1 (hARD1ΔN), which can no longer access the nucleus, resulted in cell morphology changes and cellular growth impairment. Notably, hARD1ΔN-expressing cells showed alterations in the cell cycle and the expression levels of cell cycle regulators compared to hARD1 wild-type cells. Furthermore, these effects were rescued when the nuclear import of hARD1 was restored by exogenous NLS. Our results show that hARD1 nuclear translocation mediated by NLS is required for cell cycle progression, thereby contributing to proper cell proliferation.     

MEL-28 is downstream of the Ran cycle and is required for nuclear-envelope function and chromatin maintenance

Early embryonic development depends on the faithful execution of basic cell biological processes whose coordination remains largely unknown. With a global network analysis, we found MEL-28 to be associated with two types of complexes, one implicated in nuclear-envelope function and the other in chromatin organization. Here, we show that MEL-28, a protein that shuttles between the nucleus and the kinetochore during the cell cycle, is required for the structural and functional integrity of the nuclear envelope. In addition, mel-28(RNAi) embryos exhibit defects in chromosome condensation, pronuclear migration, kinetochore assembly, and spindle assembly. This combination of mel-28(RNAi) phenotypes resemble those caused by depleting members of the Ran cycle in C. elegans, a conserved cellular signaling pathway that is required for mitotic spindle assembly, nuclear-envelope reformation after mitosis, and nucleocytoplasmic exchange (reviewed in). Although MEL-28 localization to the nuclear periphery is not dependent on nuclear pore components, it is dependent on RAN-1 and other key components of the Ran cycle. Thus, MEL-28 is downstream of the Ran cycle and is required for both proper nuclear-envelope function and chromatin maintenance.     

Loss of Acinus inhibits oligonucleosomal DNA fragmentation but not chromatin condensation during apoptosis

Chromatin condensation and oligonucleosomal DNA fragmentation are the nuclear hallmarks of apoptosis. A proteolytic fragment of the apoptotic chromatin condensation inducer in the nucleus (Acinus), which is generated by caspase cleavage, has been implicated in mediating apoptotic chromatin condensation prior to DNA fragmentation. Acinus is also involved in mRNA splicing and a component of the apoptosis and splicing-associated protein (ASAP) complex. To study the role of Acinus for apoptotic nuclear alterations, we generated stable cell lines in which Acinus isoforms were knocked down by inducible and reversible RNA interference. We show that Acinus is not required for nuclear localization and interaction of the other ASAP subunits SAP18 and RNPS1; however, knockdown of Acinus leads to a reduction in cell growth. Most strikingly, down-regulation of Acinus did not inhibit apoptotic chromatin condensation either in intact cells or in a cell-free system. In contrast, although apoptosis proceeds rapidly, analysis of nuclear DNA from apoptotic Acinus knockdown cells shows inhibition of oligonucleosomal DNA fragmentation. Our results therefore suggest that Acinus is not involved in DNA condensation but rather point to a contribution of Acinus in internucleosomal DNA cleavage during programmed cell death.     

Importin β2 Mediates the Spatio-temporal Regulation of Anillin through a Noncanonical Nuclear Localization Signal

The compartmentalization of cell cycle regulators is a common mechanism to ensure the precise temporal control of key cell cycle events. For instance, many mitotic spindle assembly factors are known to be sequestered in the nucleus prior to mitotic onset. Similarly, the essential cytokinetic factor anillin, which functions at the cell membrane to promote the physical separation of daughter cells at the end of mitosis, is sequestered in the nucleus during interphase. To address the mechanism and role of anillin targeting to the nucleus in interphase, we identified the nuclear targeting motif. Here, we show that anillin is targeted to the nucleus by importin β2 in a Ran-dependent manner through an atypical basic patch PY nuclear localization signal motif. We show that although importin β2 binding does not regulate anillin''s function in mitosis, it is required to prevent the cytosolic accumulation of anillin, which disrupts cellular architecture during interphase. The nuclear sequestration of anillin during interphase serves to restrict anillin''s function at the cell membrane to mitosis and allows anillin to be rapidly available when the nuclear envelope breaks down to remodel the cellular architecture necessary for successful cell division.     

Electron-radioautographic analysis of 3H-thymidine distribution in the cell nuclei of the Chinese hamster

Three types of the label localization in the nuclei of Chinese hamster fibroblasts, growing for 9 and 13 hours with 3H-thymidine, were detected using electron microscopic autoradiography: 1. The label is relatively evenly distributed throughout the karyoplasm. 2. Silver grains are concentrated as stripes through the nucleus; a high label density is also found in the nuclear periphery and around the nucleolus. 3. The label is mainly concentrated over the condensed chromatin adjacent to the nuclear membrane. The cells labeled in the first half of S-phase and selected with colchicine in postsynthetic phase of the 1st and the 2nd cycles are characterized by the second and third types of label distribution. In the cell nuclei fixed in the postsynthetic period of the second cycle, the label localization in stripes is discontinuous. The results indicate that during cell transition from S to G2 the newly-synthetized DNA changes its localization in the nucleus. It is suggested that the second type of label distribution depends on the interphase chromosome concentration in definite zones of the nuclear volume after S-phase termination, and the third type label localization is connected with the formation of prophase chromosomes.