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.

Nuclei contain two differentially regulated pools of diacylglycerol.

A number of recent studies have highlighted the presence of a nuclear pool of inositol lipids [1] [2] that is regulated during progression through the cell cycle [1] [3], differentiation [1] [2] and after DNA damage [2], suggesting that a number of different regulatory pathways impinge upon this pool of lipids. It has been suggested that the downstream consequence of the activation of one of these nuclear phosphoinositide (PI) regulatory pathways is the generation of nuclear diacylglycerol (DAG) [1] [3] [4], which is important in the activation of nuclear protein kinase C (PKC) [5] [6] [7]. Activation of PKC in turn appears to regulate the progression of cells through G1 and into S phase [4] and through G2 to mitosis [3] [8] [9] [10] [11]. Although the evidence is enticing, there is as yet no direct demonstration that nuclear PIs can be hydrolysed to generate nuclear DAG. Previous data in murine erythroleukemia (MEL) cells have suggested that nuclear phosphoinositidase Cbeta1 (PIC-beta1) activity is important in the generation of nuclear DAG. Here, we demonstrate that the molecular species of nuclear DAG bears little resemblance to the PI pool and is unlikely to be generated directly by hydrolysis of these inositol lipids. Further, we show that there are in fact two distinct subnuclear pools of DAG; one that is highly disaturated and mono-unsaturated (representing more than 90% of the total nuclear DAG) and one that is highly polyunsaturated and is likely to be derived from the hydrolysis of PI. Analysis of these pools, either after differentiation or during cell-cycle progression, suggests that the pools are independently regulated, possibly by the regulation of two different nuclear phospholipase Cs (PLCs).

Regulation of type IIalpha phosphatidylinositol phosphate kinase localisation by the protein kinase CK2.

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIalpha PIP kinase at a single site unique to that isoform - Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.

A novel pathway of cellular phosphatidylinositol(3,4,5)-trisphosphate synthesis is regulated by oxidative stress.

BACKGROUND: Phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is a key second messenger found ubiquitously in higher eukaryotic cells. The activation of Class I phosphoinositide 3-kinases and the subsequent production of PtdIns(3,4,5)P(3) is an important cell signaling event that has been causally linked to the activation of a variety of downstream cellular processes, such as cell migration and proliferation. Although numerous proteins regulating a variety of biological pathways have been shown to bind PtdIns(3,4,5)P(3), there are no data to demonstrate multiple mechanisms for PtdIns(3,4,5)P(3) synthesis in vivo. RESULTS: In this study, we demonstrate an alternative pathway for the in vivo production of PtdIns(3,4,5)P(3) mediated by the action of murine Type Ialpha phosphatidylinositol 4-phosphate 5-kinase (Type Ialpha PIPkinase), an enzyme best characterized as regulating cellular PtdIns(4,5)P(2) levels. Analysis of this novel pathway of PtdIns(3,4,5)P(3) synthesis in cellular membranes leads us to conclude that in vivo, Type Ialpha PIPkinase also acts as a PtdIns(3,4)P(2) 5-kinase. We demonstrate for the first time that cells actually contain an endogenous PtdIns(3,4)P(2) 5-kinase, and that during oxidative stress, this enzyme is responsible for PtdIns(3,4,5)P(3) synthesis. Furthermore, we demonstrate that by upregulating the H(2)O(2)-induced PtdIns(3,4,5)P(3) levels using overexpression studies, the endogenous PtdIns(3,4)P(2) 5-kinase is likely to be Type Ialpha PIPkinase. CONCLUSIONS: We describe for the first time a novel in vivo activity for Type Ialpha PIPkinase, and a novel pathway for the in vivo synthesis of functional PtdIns(3,4,5)P(3), a key lipid second messenger regulating a number of diverse cellular processes.

Multivesicular body morphogenesis requires phosphatidyl-inositol 3-kinase activity.

Multivesicular bodies are endocytic compartments containing multiple small vesicles that originate from the invagination and 'pinching off' of the limiting membrane into the luminal space [1] [2] [3]. The molecular mechanisms responsible for the formation of these compartments are unknown. In the human melanoma cell line Mel JuSo, newly synthesised major histocompatibility complex (MHC) class II molecules accumulate in multivesicular early lysosomes [4]. The phosphatidylinositol (PI) 3-kinase inhibitor wortmannin induced the transient vacuolation of early MHC class II compartments, but also of early and late endosomes. We demonstrate that endocytic membrane influx is required for the wortmannin-induced swelling of vesicles. The wortmannin-induced vacuoles contained a reduced number of intraluminal vesicles that were linked to the limiting membrane by membraneous connections. These data suggest that wortmannin inhibits the invagination and/or pinching off of intraluminal vesicles and provide evidence of a role for PI 3-kinase in multivesicular body morphogenesis. We propose that the wortmannin-induced vacuolation occurs as a result of the inability of multivesicular bodies to store endocytosed membranes as intraluminal vesicles thereby causing the formation of large 'empty' vacuoles.

Phospholipases in the nucleus.

There is a well established role for various phospholipases involved in the production of intracellular signals at the plasma membrane. In contrast much less is known of their role in other intracellular compartments, however, emerging evidence would suggest that some of these enzymes are also involved in the production of signals within the nucleus. Translocation to and activation of protein kinase C (PKC) within the nucleus has been suggested to be important in a number of diverse cellular processes suggesting the requirement for the intranuclear production of diacylglycerol (DAG), a known physiological activator of this enzyme. As the activation of a number of phospholipases leads to the production of DAG this review will consider the notion that these enzymes are present within the nucleus and that their activities can be stimulated to produce this important regulator of PKC.

Molecular species analysis of 1,2-diacylglycerols and phosphatidic acid formed during bombesin stimulation of Swiss 3T3 cells.

Swiss 3T3 cells were labelled with [3H]glycerol and stimulated with bombesin over a time course of 20 min. The individual 1,2-diacylglycerols produced were quantified by acetylation followed by analysis by HPLC and argentation chromatography. The major phospholipids and phosphatidic acid were acetolysed and then analysed in the same manner. The data show that even at an early time of stimulation (30 s), stimulated diacylglycerol formation comes from at least two sources--phosphoinositides and phosphatidylcholine.

Phospholipid signalling in the nucleus. Een DAG uit het leven van de inositide signalering in de nucleus.

Diverse methodologies, ranging from activity measurements in various nuclear subfractions to electron microscopy, have been used to demonstrate and establish that many of the key lipids and enzymes responsible for the metabolism of inositol lipids are resident in nuclei. PtdIns(4)P, PtdIns(4,5)P2 and PtdOH are all present in nuclei, as well as the corresponding enzyme activities required to synthesise and metabolise these compounds. In addition other non-inositol containing phospholipids such as phosphatidylcholine constitute a significant percentage of the total nuclear phospholipid content. We feel that it is pertinent to include this lipid in our discussion as it provides an alternative source of 1, 2-diacylglycerol (DAG) in addition to the hydrolysis of PtdIns(4, 5)P2. We discuss at length data related to the sources and possible consequences of nuclear DAG production as this lipid appears to be increasingly central to a number of general physiological functions. Data relating to the existence of alternative pathways of inositol phospholipid synthesis, the role of 3-phosphorylated inositol lipids and lipid compartmentalisation and transport are reviewed. The field has also expanded to a point where we can now also begin to address what role these lipids play in cellular proliferation and differentiation and hopefully provide avenues for further research.

The nuclear phosphoinositide cycle--does it play a role in nuclear Ca2+ homoeostasis?

The probable answer to this question is no. Much of the current evidence summarised elsewhere in this issue points to nuclear Ca2+ changes changing in response to cytosolic Ca2+, with little evidence for an independently controlled nuclear Ca2+ homeostasis. There are InsP3 receptors in the nuclear membrane, and it is possible that during nuclear membrane assembly the InsP3 acting on these (Sullivan and Wilson, this issue) is formed by an inositide cycle located on the assembling nuclear skeleton. But our current experimental data suggest that when the nucleus is intact, InsP3 generated by this cycle would have to exit through the nuclear pores to act on any known InsP3 receptors. Thus the nuclear inositide cycle appears more likely to serve to generate diacylglycerol to activate protein kinase C, and/or to generate inositol phosphates such as InsP2, which may have distinct intranuclear functions.

Cloning and characterisation of two new cDNAs encoding murine triple LIM domains.

We have cloned and sequenced two new cDNAs that code for proteins carrying the related triple LIM domains (acronym of Lin-11, Isl-1, Mec-3) proteins. These LIM domains show good agreement to the LIM domain consensus sequence, but also exhibit some novel variations. The 1.36-and 2.8-kb cDNAs are probably splice variants of one gene and code for 42- and 50-kDa proteins, respectively. The larger transcript has a 900-nucleotide (nt) 3' untranslated region (UTR). High levels of the 2.8-kb transcript can be detected in many tissues, and all tissues show some level of expression of both transcripts, the larger transcript being more abundant. In adult testis there are very high levels of the 1.36-kb transcript and moderate levels of the 2.8-kb transcript. The wide tissue distribution and high levels of expression suggest an important role for these proteins in cellular function.

DNA-dependent protein kinase and related proteins.

The DNA-dependent protein kinase (DNA-PK) is a nuclear protein serine/threonine kinase that must bind to DNA double-strand breaks to be active. We and others have shown that it is a multiprotein complex comprising an approx. 465 kDa catalytic subunit (DNA-PKcs) and a DNA-binding component, Ku. Notably, cells defective in DNA-PK are hypersensitive to ionizing radiation. Thus X-ray-sensitive hamster xrs-6 cells are mutated in Ku, and rodent V3 cells and cells of the severe combined immune-deficient (Scid) mouse lack a functional DNA-PKcs. Cloning of the DNA-PKcs cDNA revealed that it falls into the phosphatidylinositol (PI) 3-kinase family of proteins. However, biochemical assays indicate that DNA-PK contains no intrinsic lipid kinase activity, but is instead a serine/threonine kinase. We have also found that DNA-PK activity can be inhibited by the PI 3-kinase inhibitors wortmannin and LY294002. Consistent with its proposed role in genome surveillance and the detection of DNA damage, DNA-PKcs is most similar to a subset of proteins involved in cell-cycle checkpoint control and signalling of DNA damage. Furthermore, the recent cloning of the gene mutated in ataxia-telangiectasia (A-T) patients, named ATM (A-T mutated), has revealed that the product of this gene is also a PI 3-kinase family member and is related to DNA-PKcs. Although much is known about the clinical symptoms and cellular phenotypes that arise from disruption of the A-T gene, little is known about the biochemical action of ATM in response to DNA damage. Given its sequence similarity with DNA-PKcs, we speculate that ATM may function in a manner similar to DNA-PK.

Constitutive active GTPases Rac and Cdc42 are associated with endoreplication in PAE cells.

The Rho-like guanine triphosphate (GTP)ases become activated by extracellular ligands and regulate a wide variety of biological processes, including cell motility, spreading of cells, cytoskeletal organisation and transcriptional activity. We studied the effect of expression of WtRac and Cdc42 and of their constitutive active V12 variants on cell cycle transition using the isopropylthiogalactoside (IPTG) inducible Rac and Cdc42 transfectants of porcine aortic endothelial (PAE) cells. Expression of V12Rac or V12Cdc42 resulted initially in an enrichment of cells in G2/M, followed by the appearance of multinucleated cells with some of the nuclei still being able to incorporate bromodeoxyuridine (BrdU). By fluorescent activated cell sorter (FACS) analysis, these cells appeared as polyploid cells. Prolonged activation of V12Rac or V12Cdc42 resulted in genomic instability and these cells finally detached from the culture plate. These findings indicate that induction of the constitutive active V12 forms of Rac and Cdc42 results in 'mitotic slippage', where endoreplication takes place irrespective of the exit from cytokinesis.

Isoprenaline-induced and constitutive members of a proline-rich protein sub-group from mouse parotid glands studied with monoclonal antibody NAL1.

Members of the proline-rich protein (PRP) family of mouse parotid glands were analysed before and after stimulation with the beta-agonist isoprenaline by using a monoclonal antibody raised against the induced PRP A3(0) (GP-27). Antibody NAL1 reacted strongly with isoprenaline-induced B-type PRP precursors and their salivary counterparts, but not against the A-type PRPs A1(0) (Gp-66) and A2(0) (GP-45) or human salivary proteins, and it is likely that NAL1 recognizes a proline-rich repeat variant unique to this group of rodent PRPs. PRP-related antigens were observed in the parotid glands (N1(0) and N2(0] and saliva of normal mice. The antigens were located immunocytochemically in secretory granules of parotid acinar cells of both normal and isoprenaline-stimulated mice. The total amount of PRP antigens increased 16-fold from 2.5 to 40% of parotid protein after 10 days of isoprenaline treatment, as estimated by enzyme-linked immunosorbent assay. Immunoblotting showed that new PRP species appeared during the period of increase. After treatment with isoprenaline, B-type PRPs appeared first, followed by A3(0) and another member of the family. These results show that the mouse PRP family is larger than previously thought and can be divided immunologically into sub-groups. That a subset of PRPs are produced in the normal mouse indicates that there is differential beta-adrenergic regulation within the family, and also has implications for the role of PRPs in the normal maintenance of healthy dentition and other processes.

Phospholipids in the nucleus--metabolism and possible functions.

Most of the phospholipids in the nuclear envelope are contained in the double nuclear membrane, and this has an active lipid metabolism consistent with its origins as a component of the endoplasmic reticular system. However, even after removal of the nuclear membrane with detergents, some phospholipids, mostly of unknown location and function, remain. Amongst these are all of the components of what appears to be a nuclear polyphosphoinositide signalling system, distinct from the well-established inositide pathway found in the plasma membrane. The consequences for nuclear function of the activation of these two inositide pathways are discussed, with a detailed consideration of proposed intranuclear functions for protein kinase C, and the maintenance of nuclear Ca2+ homoeostasis.

The polyphosphoinositide cycle exists in the nuclei of Swiss 3T3 cells under the control of a receptor (for IGF-I) in the plasma membrane, and stimulation of the cycle increases nuclear diacylglycerol and apparently induces translocation of protein kinase C to the nucleus.

When Swiss 3T3 cells are treated with Insulin-like Growth Factor I, a rapid decrease in the mass of polyphosphoinositol lipids (phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate) occurs within the nuclei, with a concomitant increase in nuclear diacylglycerol and translocation of protein kinase C to the nuclear region. This is in contrast to the effects of the regulatory peptide, bombesin, which causes similar inositol lipid changes in the plasma membrane, has no effect on nuclear inositide levels and causes a translocation of protein kinase C to post-nuclear membranes. These results suggest the existence of a discrete nuclear polyphosphoinositide signalling system entirely distinct from the well-known plasma membrane-located system, which is under regulatory control by cell surface-located receptors.

Purification and characterization of phosphatidylinositol 4-phosphate 5-kinases.

We detail the purification and characterization of three distinct isoforms of PtdIns4P 5-kinase present in bovine brain. One of these, PtdIns4P 5-kinase C, was purified to apparent homogeneity, and SDS/PAGE analysis demonstrated a single polypeptide and molecular mass 53 KDa. These three isoforms were shown to differ in their kinetic properties, and immunological characterization with an antibody raised to PtdIns4P 5-kinase C demonstrated that this isoform was unrelated to the other two. Furthermore, PtdIns4P 5-kinase C was shown to be the bovine brain homologue of the Type II PtdIns4P 5-kinase previously purified from human erythrocytes [Bazenet, Ruano, Brockman & Anderson (1990) J. Biol. Chem. 265, 18012-18022].

Aggregation-dependent, integrin-mediated increases in cytoskeletally associated PtdInsP2 (4,5) levels in human platelets are controlled by translocation of PtdIns 4-P 5-kinase C to the cytoskeleton.

Thrombin-stimulated aggregation of human platelets promotes an increase in the phosphatidylinositol 4-phosphate (PtdIns 4-P) 5-kinase (PIPkin) activity in the cytoskeleton. This phenomenon is associated with translocation of PIPkin isoform C to the cytoskeleton and with an increase in the amount of phosphatidylinositol bisphosphate (PtdInsP2) bound to the cytoskeletal pellet. All three of these effects are prevented if the platelets are not stirred or if RGD-containing peptides are present, demonstrating that they require integrin activation. All three are also abolished by pretreatment with okadaic acid, which also prevents the aggregation-dependent translocation of pp60(c-src) to the cytoskeleton. The results point to the existence of a cytoskeletally associated PtdInsP2 pool under the control of integrin-mediated signals that act via PIPkin C and suggest that a common, okadaic acid-sensitive mechanism may underlie the aggregation-dependent translocation of certain signalling molecules to the platelet cytoskeleton.

Regulation of PtdIns4P 5-kinase C by thrombin-stimulated changes in its phosphorylation state in human platelets.

PtdIns(4,5)P2 production by the enzyme PtdIns4P 5-kinase C (PIPkin C) was examined in thrombin-stimulated human platelets. Thrombin caused a rapid, transient 2-3-fold increase in PIPkin activity and a transient net dephosphorylation of the enzyme. PIPkin C was phosphorylated on serine and threonine residues in unstimulated platelets; no evidence for tyrosine phosphorylation was found. The phosphatase inhibitor okadaic acid promoted PIPkin C hyperphosphorylation and a concomitant marked inhibition of its activity in immunoprecipitates. Activity was restored by treatment with alkaline phosphatase, suggesting the existence of an inhibitory phosphorylation site. In support of this idea, alkaline phosphatase treatment of PIPkin C immunoprecipitated from unstimulated platelets caused a modest (1.6-fold) but significant activation of the enzyme. However, alkaline phosphatase treatment of PIPkin C immunoprecipitated from thrombin-stimulated platelets caused a decrease in activity to approximately the same levels, suggesting that the phosphorylation of PIPkin C also contributes to the observed stimulation. Two-dimensional phosphopeptide mapping of immunoprecipitated PIPkin C revealed that the enzyme is multiply phosphorylated and that, whereas some phosphopeptides are indeed lost on stimulation, consistent with the net dephosphorylation of the enzyme, at least two novel sites become phosphorylated. This suggests that thrombin causes complex changes in the phosphorylation state of PIPkin C, one consequence of which is its activation.

Nuclear diacylglycerol is increased during cell proliferation in vivo.

Highly purified nuclei were prepared from livers and kidneys of rats undergoing compensatory hepatic or renal growth, the former being predominantly by cellular proliferation, and the latter mostly by cellular enlargement. In liver, an increase in nuclear diacylglycerol (DAG) concentration occurred between 16 and 30 h, peaking at around 20 h. At the peak of nuclear DAG production a specific translocation of protein kinase C to the nucleus could be detected; no such changes occurred in kidney. There was no detectable change in whole-cell DAG levels in liver, and the increase in DAG was only measurable in nuclei freed of their nuclear membrane. Overall, these results suggest that there is a stimulation of intranuclear DAG production, possibly through the activation of an inositide cycle [Divecha, Banfic and Irvine (1991) EMBO J. 10, 3207-3214] during cell proliferation in vivo.