Nuclear Phosphoinositides: Their Regulation and Roles in Nuclear Functions.

Polyphosphoinositides (PPIns) are a family of seven lipid messengers that regulate a vast array of signalling pathways to control cell proliferation, migration, survival and differentiation. PPIns are differentially present in various sub-cellular compartments and, through the recruitment and regulation of specific proteins, are key regulators of compartment identity and function. Phosphoinositides and the enzymes that synthesise and degrade them are also present in the nuclear membrane and in nuclear membraneless compartments such as nuclear speckles. Here we discuss how PPIns in the nucleus are modulated in response to external cues and how they function to control downstream signalling. Finally we suggest a role for nuclear PPIns in liquid phase separations that are involved in the formation of membraneless compartments within the nucleus.

Multiple forms of PKR present in the nuclei of acute leukemia cells represent an active kinase that is responsive to stress.

A number of cancers possess constitutive activity of the dsRNA-dependent kinase, PKR. Inhibition of PKR in these cancers leads to tumor cell death. We recently reported the increased presence of PKR phosphorylated on Thr451 (p-T451 PKR) in clinical samples from myelodysplastic syndrome (MDS) patients and acute leukemia cell lines. Whereas p-T451 PKR in low-risk patient samples or PTEN-positive acute leukemia cell lines was mostly cytoplasmic, in high-risk patient samples and acute leukemia cell lines deficient in PTEN, p-T451 PKR was mainly nuclear. As nuclear activity of PKR has not been previously characterized, we examined the status of nuclear PKR in acute leukemia cell lines. Using antibodies to N-terminus, C-terminus and the kinase domain in conjunction with a proteomics approach, we found that PKR exists in diverse molecular weight forms in the nucleus. Analysis of PKR transcripts by reverse transcriptase-PCR, and PKR-derived peptides by MS/MS revealed that these forms were the result of post-translational modifications (PTMs). Biochemical analysis demonstrated that nuclear PKR is an active kinase that can respond to stress. Given the association of PKR with PTEN and the Fanconi complex, these results indicate that PKR likely has other previously unrecognized roles in nuclear signaling that may contribute to leukemic development.

Nuclear phosphoinositide specific phospholipase C (PI-PLC)-beta 1: a central intermediary in nuclear lipid-dependent signal transduction.

Several studies have demonstrated the existence of an autonomous intranuclear phospho-inositide cycle that involves the activation of nuclear PI-PLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PI-PLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-beta1. Nuclear PI-PLC-beta1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-beta1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-beta1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.

Nuclear protein kinase C isoforms: key players in multiple cell functions?

Protein kinase C (PKC) isozymes are a family of serine/threonine protein kinases categorized into three subfamilies: classical, novel, and atypical. PKC isozymes, whose expression is cell type-specific and developmentally regulated, are key transducers in many agonist-induced signaling cascades. To date at least 10 different PKC isotypes have been identified and are believed to play distinct regulatory roles. PKC isoforms are catalytically activated by several lipid cofactors, including diacylglycerol. PKC is thought to reside in the cytoplasm in an inactive conformation and to translocate to the plasma membrane or cytoplasmic organelles upon cell activation by different stimuli. However, a sizable body of evidence collected over the last 15 years has shown PKC to be capable of translocating to the nucleus. Furthermore, PKC isoforms can reside within the nucleus. Studies from independent laboratories have to led to the identification of several nuclear proteins which act as PKC substrates as well as to the characterization of some nuclear PKC-binding proteins which may be of fundamental importance for finely tuning PKC function in this peculiar cell microenvironment. Most likely, nuclear PKC isozymes are involved in the regulation of several important biological processes such as cell proliferation and differentiation, neoplastic transformation, and apoptosis. In this review, we shall summarize the most intriguing evidence about the roles played by nuclear PKC isozymes.

Insulin selectively stimulates nuclear phosphoinositide-specific phospholipase C (PI-PLC) beta1 activity through a mitogen-activated protein (MAP) kinase-dependent serine phosphorylation.

Using NIH 3T3 cells, we have investigated nuclear phosphoinositide metabolism in response to insulin, a molecule which acts as a proliferating factor for this cell line and which is known as a powerful activator of the mitogen-activated protein (MAP) kinase pathway. Insulin stimulated inositol lipid metabolism in the nucleus, as demonstrated by measurement of the diacylglycerol mass produced in vivo and by in vitro nuclear phosphoinositide-specific phospholipase C (PI-PLC) activity assay. Despite the fact that nuclei of NIH 3T3 cells contained all of the four isozymes of the beta family of PI-PLC (i.e. beta1, beta2, beta3, and beta4), insulin only activated the beta1 isoform. Insulin also induced nuclear translocation of MAP kinase, as demonstrated by Western blotting analysis, enzyme activity assays, and immunofluorescence staining, and this translocation was blocked by the specific MAP kinase kinase inhibitor PD98059. By means of both a monoclonal antibody recognizing phosphoserine and in vivo labeling with [(32)P]orthophosphate, we ascertained that nuclear PI-PLC-beta1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI-PLC-beta1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI-PLC-beta1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway.

A role for nuclear phospholipase Cbeta 1 in cell cycle control.

Phosphoinositide signaling resides in the nucleus, and among the enzymes of the cycle, phospholipase C (PLC) appears as the key element both in Saccharomyces cerevisiae and in mammalian cells. The yeast PLC pathway produces multiple inositol polyphosphates that modulate distinct nuclear processes. The mammalian PLCbeta(1), which localizes in the nucleus, is activated in insulin-like growth factor 1-mediated mitogenesis and undergoes down-regulation during murine erythroleukemia differentiation. PLCbeta(1) exists as two polypeptides of 150 and 140 kDa generated from a single gene by alternative RNA splicing, both of them containing in the COOH-terminal tail a cluster of lysine residues responsible for nuclear localization. These clues prompted us to try to establish the critical nuclear target(s) of PLCbeta(1) subtypes in the control of cell cycle progression. The results reveal that the two subtypes of PLCbeta(1) that localize in the nucleus induce cell cycle progression in Friend erythroleukemia cells. In fact when they are overexpressed in the nucleus, cyclin D3, along with its kinase (cdk4) but not cyclin E is overexpressed even though cells are serum-starved. As a consequence of this enforced expression, retinoblastoma protein is phosphorylated and E2F-1 transcription factor is activated as well. On the whole the results reveal a direct effect of nuclear PLCbeta(1) signaling in G(1) progression by means of a specific target, i.e. cyclin D3/cdk4.

Identification and chromosomal localisation by fluorescence in situ hybridisation of human gene of phosphoinositide-specific phospholipase C beta(1).

Members of phosphoinositide-specific phospholipase C (PLC) families are central intermediary in signal transduction in response to the occupancy of receptors by many growth factors. Among PLC isoforms, the type beta(1) is of particular interest because of its reported nuclear localisation in addition to its presence at the plasma membrane. It has been previously shown that both the stimulation and the inhibition of the nuclear PLCbeta(1) under different stimuli implicate PLCbeta(1) as an important enzyme for mitogen-activated cell growth as well as for murine erythroleukaemia cell differentiation. The above findings hinting at a direct involvement of PLCbeta(1) in controlling the cell cycle in rodent cells, and the previously reported mapping of its gene in rat chromosome band 3q35-36, a region frequently rearranged in rat tumours induced by chemical carcinogenesis, prompted us to identify its human homologue. By screening a human foetal brain cDNA library with the rat PLCbeta(1) cDNA probe, we have identified a clone homologous to a sequence in gene bank called KIAA 0581, which encodes a large part of the human PLCbeta(1). By using this human cDNA in fluorescence in situ hybridisation on human metaphases, it has been possible to map human PLCbeta(1) on chromosome 20p12, confirming the synteny between rat chromosome 3 and human chromosome 20 and providing a novel locus of homology between bands q35-36 in rat and p12 in man. Since band 20p12 has been recently reported amplified and/or deleted in several solid tumours, the identification and chromosome mapping of human PLCbeta(1) could pave the way for further investigations on the role exerted both in normal human cells and in human tumours by PLCbeta(1), which has been shown to behave as a key signalling intermediate in the control of the cell cycle.

Inhibition of phosphoinositide 3-kinase impairs pre-commitment cell cycle traverse and prevents differentiation in erythroleukaemia cells.

During the early hours after exposure to differentiation inducing agents, Friend erythroleukaemia cells undergo alterations which commit them to cessation of growth and development of the characteristics of differentiation. Our current experiments have compared the expression and activity of phosphoinositide 3-kinase (PI 3-kinase) in control cells with cells undergoing differentiation which has been induced by dimethyl sulfoxide (DMSO). When the cultures were initiated with stationary phase cells and DMSO was added at the time of seeding, PI 3-kinase activity was stimulated in both treated and control cells during the first 3 h from seeding. This event appears to be a rate limiting step in commitment since pretreatment of cells with 10 microM LY294002 or down-regulation of p85 expression prior to adding DMSO completely prevents commitment to erythropoiesis. Accordingly, PI 3-kinase inhibition during the commitment period prevents DNA-binding of the transcription factor GATA-1, essential for erythroid differentiation. However, once cells are committed to differentiate, PI 3-kinase activity and expression dramatically decreases along with the differentiation programme, to become barely detectable after 96 h. Remarkably, LY294002 treatment leads to accumulation of cell in G1 phase and prevents DMSO-dependent cyclin D3 induction. Based on these data, we suggest that PI 3-kinase is rate limiting for the completion of the first round cycle of cell division required for initiation of erythrocytic differentiation. On the other hand, the late decrease of PI 3-kinase associated with the differentiation process seems to be part of the programmed shut off of genes not needed in mature erythrocytes.

Phosphatidylinositol 3-kinase translocation to the nucleus is induced by interleukin 1 and prevented by mutation of interleukin 1 receptor in human osteosarcoma Saos-2 cells.

Although interleukin 1 (IL-1) functions have been extensively characterized, the mechanisms by which IL-1 signals are transduced from the plasma membrane to the nucleus are less known. Recent evidence indicates that phosphatidylinositol 3-kinase (PI3-kinase) could be activated by a direct association with the activated IL-1 receptor. In this study we analyzed the effects of IL-1 on the intracellular distribution of PI3-kinase in wild-type Saos-2 human osteosarcoma cells, and in cell clones overexpressing type I IL-1 receptor (IL-1RI). PI3-kinase intracellular distribution displays two distinct patterns. In quiescent cells, PI3-kinase is distributed through the cytoplasm, although a portion is present in the nucleus; following stimulation with IL-1, PI3-kinase is redistributed, increasing in the nuclear compartment. Both immunoblotting and immunofluorescence data indicate that IL-1 causes a rapid and transient translocation of PI3-kinase from the cytoplasm to the nucleus. This phenomenon is prevented by PI3-kinase inhibitors, suggesting that the maintenance of PI3-kinase activity is essential for IL-1-induced translocation. Indeed, in cell clones stably transfected with Y479F receptor mutant, in which the binding of the enzyme to the activated receptor is blocked, IL-1-induced PI3-kinase translocation to the nucleus is completely prevented. These data suggest that PI3-kinase translocation to the nucleus upon IL-1R activation is an early event in IL-1 signaling mechanism, and may be involved in transcriptional activation.

Nuclear phospholipase C: a novel aspect of phosphoinositide signalling.

The role of polyphosphoinositides in cellular signalling is well known and recently it has also been shown that the nucleus is a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. It has been demonstrated that phospholipase C specific for inositol lipids (PLC) is one of the main steps of the inositol lipid cycle. The PLC beta family, and especially type beta 1, has given rise to considerable interest since, due to their common COOH-terminus they show nuclear localisation in addition to that at the plasma membrane. It is well established that an autonomous intranuclear inositide cycle exists, and that this cycle is endowed with conventional lipid kinases, phosphatases and PLCs. Among this latter the beta 1 type undergoes stimulation or inhibition under different stimuli and this implicates the beta 1 isoform as a key enzyme for mitogen-activated cell growth as well as for differentiation. Indeed, both the overexpression and the down-regulation of PLC beta 1, by means of antisense mRNA, have demonstrated that PLC plays a role in the nuclear compartment.

Phosphatidylinositol 3-kinase is recruited to a specific site in the activated IL-1 receptor I.

Interleukin 1 (IL-1) delivers a stimulatory signal which increases the expression of a set of genes by modulating the transcription factor NF-kappaB. The IL-1 receptors are transmembrane glycoproteins which lack a catalytic domain. The C-terminal portion of the type I IL-1 receptor (IL-IRI) is essential for IL-1 signalling and for IL-1 dependent activation of NF-kappaB. This portion contains a putative phosphatidylinositol 3-kinase (PI 3-kinase) binding domain (Tyr-E-X-Met), which is highly conserved between the human, mouse and chicken sequences, as well as the related cytoplasmic domain of the Drosophila receptor Toll. This observation prompted us to investigate the role of PI 3-kinase in IL-1 signalling. Here we report evidence that PI 3-kinase is recruited by the activated IL-IRI, causing rapid and transient activation of PI 3-kinase. We also show that the receptor is tyrosine phosphorylated in response to IL-1. Expression of a receptor mutant lacking the putative binding site for p85 demonstrates that Tyr479 in the receptor cytoplasmic domain is essential for PI 3-kinase activation by IL-1. Our results indicate that PI 3-kinase is likely to be an important mediator of some IL-1 effects, providing docking sites for additional signalling molecules.

Nuclear but not cytoplasmic phospholipase C beta 1 inhibits differentiation of erythroleukemia cells.

A body of evidence has shown the existence of a nuclear phosphoinositide cycle in different cell types. The cycle is endowed with kinases as well as phosphatases and phospholipase C (PLC). Among the PLC isozymes, the beta family is characterized by a long COOH-terminal tail that contains a cluster of lysine residues responsible for nuclear localization. Indeed, PLC beta 1 is the major isoform that has been detected in the nucleus of several cells. This isoform is activated by insulin-like growth factor I, and when this isoform is lacking, as a result of gene ablation, the onset of DNA synthesis induced by this hormone is abolished. On the contrary, PLC beta 1 is down-regulated during the erythroid differentiation of Friend erythroleukemia cells. A key question is how PLC beta 1 signaling at the nucleus fits into the erythroid differentiation program of Friend erythroleukemia cells, and whether PLC beta 1 signaling activity is directly responsible for the maintenance of the undifferentiated state of erythroleukemia cells. Here we present evidence that nuclear PLC beta 1 but not the isoform located at the plasma membrane is directly involved in maintaining the undifferentiated state of Friend erythroleukemia cells. Indeed, when wild-type PLC beta 1 is overexpressed in these cells, differentiation in response to DMSO is inhibited in that the expression of beta-globin is almost completely abolished, whereas when a mutant lacking the ability to localize to the nucleus is expressed, the cells differentiate, and the expression of beta-globin is the same as in wild-type cells.

Essential role for nuclear phospholipase C beta1 in insulin-like growth factor I-induced mitogenesis.

The nucleus has been shown to be a site for the inositol lipid cycle that can be affected by treatment of quiescent cells with growth factors such as insulin-like growth factor I (IGF-I). Indeed, the exposure of Swiss 3T3 cells to IGF-I results in a rapid and transient increase in nuclear phospholipase C (PLC) beta1 activity. In addition, several other reports have shown the involvement of PLC beta1 in nuclear signaling in different cell types. Although the demonstration of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate hydrolysis by nuclear PLC beta1 established the existence of nuclear PLC signaling, the significance of this autonomous pathway in the nucleus has yet to be thoroughly clarified. By inducing both the inhibition of PLC beta1 expression by antisense RNA and its overexpression, we show that this nuclear PLC is essential for the onset of DNA synthesis following IGF-I stimulation of quiescent Swiss 3T3 cells.

Inhibition of the expression of ornithine decarboxylase and c-Myc by cell-permeant ceramide in difluoromethylornithine-resistant leukaemia cells.

Ceramide has emerged as a novel lipid mediator in cell growth and apoptosis. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, the cell-permeant analogues of ceramide N-acetylsphingosine (C2-ceramide) and N-hexanoylsphingosine (C6-ceramide) inhibited the induction of ornithine decarboxylase (ODC) activity with IC50 of 8.3 and 1.5 microM respectively. This effect was strictly related to the ability to inhibit cell growth and [3H]thymidine incorporation. The suppression of cell growth was also associated with apoptosis. The addition of bacterial sphingomyelinase resulted in a significant, but limited, reduction of ODC induction and [3H]thymidine incorporation. Bacterial lipopolysaccharide, which may act as a ceramide analogue, also inhibited the induction of the enzyme. Moreover, C6-ceramide largely prevented the accumulation of ODC mRNA and its precursor, ODC heterogeneous nuclear RNA, that accompanied the induction of ODC activity. A slight increase in ODC turnover was also observed. The DNA-binding activity of some transcription factors known to bind and transactivate the ODC gene was investigated by gel mobility-shift assay under the same experimental conditions. However, only the binding of Myc/Max was negatively affected by the treatment with C6-ceramide. Furthermore, the amount of immunoreactive c-Myc, which increased after stimulation of the cells to growth, was strongly reduced by C6-ceramide. These results suggest that the inhibition of c-Myc and ODC expression may be early events in the response of leukaemia cells to ceramide.

Control of expression of PLCbeta1 by LAC repressor system: relationship between nuclear PLCbeta1 overexpression and growth factor stimulation.

Swiss 3T3 cells have a nuclear phosphoinositide signalling system which is under the control of insulin-like growth factor I (IGF-I) and acts separately from that at the plasma membrane. By using the Lac repressor system we were able both to obtain the inducible overexpression of phospholipase C beta1 (PLC beta1) and to determine its subcellular localisation and partitioning. Moreover, by comparing the level of expression at the nucleus and the percentage of cells actively incorporating bromodeoxyuridine (BrdU) in S phase it has strengthened the issue of the importance of this PLC in the onset of DNA synthesis mediated by IGF-I. In addition, this system appears to be a very powerful tool for further analysis of the downstream events following the activation of nuclear PLC beta1.

Interleukin-1 alpha induces variations of the intranuclear amount of phosphatidylinositol 4,5-bisphosphate and phospholipase C beta 1 in human osteosarcoma Saos-2 cells.

Some key elements of signal transduction have been identified within the nucleus and demonstrated to be responsive to specific agonists in numerous cell types. In particular, mitogenic stimuli have been reported to induce a transient increase of the nuclear phospholipase C beta 1 activity, causing the release of inositide-derived second messengers, whereas differentiating stimuli induced a decrease of the enzyme activity and an increase of nuclear phosphatidylinositol 4,5-bisphosphate (PIP2). Recently, we reported evidence, in human osteosarcoma Saos-2 cell lines, on the presence of specific nuclear phospholipase C isoforms and on the activation of phospholipase C beta 1 in the nucleus following the exposure to interleukin-1 alpha. In this study we report immunocytochemical ultrastructural evidence on quantitative variations of PIP2 and phospholipase C beta 1 amounts in the nucleus of Saos-2 cells at different times of exposure to interleukin-1 alpha. After short periods of culture in the presence of the agonist, the intranuclear amount of PIP2 is decreased, while a translocation of phospholipase C beta 1 occurs from the cytoplasm to the nucleus, in correspondence with the increased hydrolyzing activity of the enzyme. After longer periods of incubation with interleukin-1 alpha, on the other hand, the intranuclear amount of PIP2 is restored to initial level, while the amount of phospholipase C beta 1 is increased both at the nuclear and cytoplasmic level, when its activation is no longer effective. The results, compared with those obtained in other cell types responsive to given agonists, account for a cell-specific modulation of signal transduction based on polyphosphoinositide breakdown at the nuclear level.

Transfected Saos-2 cells overexpressing phosphoinositidase C beta 1 isoform accumulate it within the nucleus.

The subcellular partitioning of the phosphoinositidase C (PIC) isoforms involved in signal transduction, with the selective localization of the PIC beta 1 isoform in the nucleus, represents a crucial aspect of the complex mechanism of cell response to agonists. In order to further elucidate this phenomenon, we utilized human osteosarcoma Saos-2 cells, transfected with the cDNA for rat PIC beta 1. In the cells overexpressing this isoform, immunocytochemical analyses at the electron microscope level reveal an increased synthesis at the cytoplasm and a significant accumulation within the nucleus of the protein. Interestingly, the sites of intranuclear localization are, as in wild type cells, the interchromatin domains. These results indicate that the transfected cells maintain the capability of accumulating the enzyme within the nucleus and can be considered a model for functional studies on the nuclear signal transduction also in response to specific agonists.

The intranuclear amount of phospholipase C beta 1 decreases following cell differentiation in Friend cells, whereas gamma 1 isoform is not affected.

The existence of a signal transduction system in the nucleus, based on polyphosphoinositide breakdown mediated by specific phosphoinositidases (PLC), has been widely documented. In different cell systems, nuclear PLCs can be modulated, in response to agonists, either by enhancing or by down-regulating their activity, thus leading to DNA replication or to cell differentiation. Friend cells, induced to erythroid differentiation by dimethyl sulfoxide (DMSO), show a down-regulation of PLC beta 1 isoform, as indicated by the reduction of the transcription of its mRNA and of the in vitro synthesis of its translation product. The intracellular localization and the amount of different PLC isoforms have been evaluated by electron microscope immunocytochemistry. In untreated Friend cells, PLC beta 1 and gamma 1 isoforms are both present within the nucleus, whereas mainly the gamma 1 isoform is detected in the cytoplasm. The small amount of cytoplasmic PLC beta 1 is probably representative only of the newly synthesized enzyme. Quantitative immunolabeling analyses demonstrate that erythroid differentiation is associated with a significant decrease of the PLC beta 1 amount in the nucleus and with an almost complete disappearance of that isoform in the cytoplasm, whereas the PLC gamma 1 isoform is unaffected. The two PLC isoforms, moreover, appear to be differently associated with the nuclear components, PLC beta 1 being steadily bound to the inner nuclear matrix, whereas PLC gamma 1 is almost completely soluble.

Nuclear pores in the apoptotic cell.

During apoptosis, nuclear pores undergo strong modifications, which are described here in five different apoptotic models. Conventional electron microscopy, supported by freeze-fracture analysis, showed a constant migration of nuclear pores towards the diffuse chromatin areas. In contrast, dense chromatin areas appear pore-free and are frequently surrounded by strongly dilated cisternae. A possible functional significance of this pore behaviour during apoptosis is discussed.